Le complexe de ligation dans la réaction de réparation des cassures de l'ADN par recombination non homologué
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(3) Acknowledgements ~1000 days are just like a glance but have shaped my life entirely. What I experienced here in Toulouse will be engraved in my memory eternally with countless gratitude. My first thank should go to Pr. Bernard Salles who provided me opportunity at first place, accepted me to work in the group without met me and known me at all, guided me in the works and directed this thesis as well as gave me numerous helps to adapt the life here. My special thank must go to Dr. Patrick Calsou who paid too much patience to lead me in the research, shoot the troubles, discuss with me, inspire me with great interpretations, broaden my understanding, bear my awful presentations, correct my thesis, and finally give me a JBC, even not being directly responsible for my thesis training. I owe my foremost thanks to Dr. Philippe Frit who not only assisted me in all the research works, but also spent plenty of time to discuss with me, solve all my problems, expand my knowledge, interpret my results, share me with smart ideas, and most important of all, vigorously edit my thesis manuscript as well as train my defense presentation. Without his one-week intensive correction, this thesis work would be in dystocia and cannot be completed on time. I believe our Ligase IV story will deserve a good destiny. How fortunate I am in having three such incredible mentors at the same time that smoothes my doctoral training. I would also like to thank my committee members, Dr. Ming-Daw Tsai, Dr. JeanPierre de Villartay, and Pr. Hervé Prats for reading and commenting my thesis manuscript as well as providing insightful suggestion and discussion in my thesis defense. I gratefully acknowledge all the past and present members of Equipe Salles for indulging me using English as almost the only communicating tool, bearing my lousy French comprehension and assisting me when I need help. I truly thank Sébastien for all his kind and selfless helps, suggestions, and friendship. I especially thank Christine and Stéphanie for their technical supports and worm cares that makes me feel home. I’d also like to thank Fanny and Aline for giving me hands dealing with several annoying but urgent stuffs, so are other colleagues, Pr. Catherine Muller, Dr. Dennis Gomez, Marielle, Béatrice, Sandrine, Oriane, Céline, Frédérique, Karine etc. and those co-workers in IPBS ever helped me. I dedicate this work to my parents for their endless encouragements and supports; without so, I would not dare to pursuit what I have earned and achieved now. I cannot express enough sorry and thankfulness to my love, Yi-Ying Cheng who endures the days without my company, shares my pressure and tolerates my madness.. !"#$%&'(')%*+,'.
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(5) •. Résumé. Les cassures double-brin (CDB) de l’ADN sont produites lors d’événements physiologiques ou physiopathologiques comme la recombinaison V(D)J, le blocage de fourches de replication, ou bien induites par des agents physiques ou chimiques à activité clastogène. Les CDB représentent une lésion hautement toxique et sont réparées par recombinaison homologue ou par recombinaison non-homologue (NHEJ). Chez les mammifères, la voie NHEJ représente le processus majoritaire de réparation responsable de la survie cellulaire après endommagement de l’ADN. Suite à la production de CDB, par exemple par des rayonnements ionisants (RI), l’hétérodimère Ku70/80 se lie aux extrémités de la cassure et recrute la sous-unité catalytique DNAPKcs. Ce Complexe-1 ou DNA-PK (i.e Ku70/Ku80/DNA-PKcs) forme une synapse qui maintient rapprochées les extrémités de la cassure, acquiert une activité sérinethréonine kinase qui par auto-phosphorylation de la DNA-PKcs induit un changement de conformation favorisant le recrutement du 2ème complexe impliqué dans la ligation des extrémités. Ce Complexe-2 est un hétérotrimère composé de XRCC4, Ligase IV et Cernnunos-XLF. Ce travail a eu pour objectif de mieux comprendre les interactions internes et externes des partenaires du Complexe-2. Nous avons établi le domaine minimal d’interaction fonctionnelle de la Ligase IV (XIR-BRCT2) avec XRCC4 et montré que son expression cellulaire induit une sensibilisation au RI et autres agents clastogènes. Le mécanisme de sensibilisation repose sur un déplacement de la Ligase IV du Complexe2 suivie de sa dégradation aboutissant à une perte de recrutement stable du complexe sur la chromatine endommagée. En parallèle, nous avons suivi le recrutement de Cernnunos-XLF et montré qu’il était un substrat de la DNA-PK en réponse aux CDB. Bien que l’absence de Cernnunos-XLF n’affecte pas le recrutement des autres partenaires (Complexe-1 et -2) son recrutement stable aux cassures repose sur la présence de DNA-PKcs, Ligase IV et XRCC4. En conclusion, nos résultats permettent de mieux comprendre l’architecture du complexe de ligation et ouvrent la voie à la recherche de composés présentant potentiellement une activité radio- ou chimiosensibilisatrice par interférence avec le mécanisme de réparation des cassures de l’ADN par NHEJ.. 1.
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(7) •. Abstract. Functional interactions between components of the ligation complex of DNA double strand break repair by Non-Homologous End-Joining DNA double-strand breaks (DSBs) are the most lethal threats among all the DNA damages in cells. They can arise not only endogenously from normal physiological processes such as V(D)J recombination or toxic lesions like DNA replication forks collapses, but also exogenously from DNA damaging agents like ionizing radiation (IR) or radiomimetic compounds. In mammals, DSBs are mainly repaired by homologous recombination (HR) during S and G2 phases of the cell cycle when sister chromatids are available, and, more predominantly, in all the phases of cell cycle by the nonhomologous end-joining (NHEJ) pathway without any requirement for homology guidance. The NHEJ machinery is also involved in V(D)J recombination to rearrange Bcell immunoglobulin and T-cell receptor genes. Deficiency in NHEJ consequently results in hypersensitivity to IR, immunodeficiency, as well as chromosomal instability. After DSBs induction, Ku70/Ku80 heterodimer binds to free DNA ends, allowing the subsequent recruitment and activation of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). The resulting DNA-PK holoenzyme (i.e. Ku/DNA-PKcs or Complex-1) tethers two DNA termini and form the synaptic complex that may further activates DNA-PKcs by several (auto)phosphorylation events. Upon activation, Complex-1 undergoes conformational changes to accommodate the ligation complex (Complex-2) and accessory factors that make DNA ends compatible with ligation, when necessary. Complex-2 comprises XRCC4, DNA LigIV (LigIV) and the more recently identified factor Cernunnos-XLF (Cer-XLF). The three partners interact with each other and Complex-2 also binds Complex-1 and accessory factors, thus accounting for its highly efficient end-joining activity. In this work we aimed at characterizing the intimate interaction network between Complex-2 factors. We refined the previously described LigIV/XRCC4 interaction interface by structural and functional evidences. LigIV was known to bind to XRCC4 dimer central helical stalks through its inter-BRCT region containing the so-called XRCC4-Interacting Region (XIR). We found that the XIR required additional stabilizing interaction between the carboxy-terminal BRCT domain of LigIV and XRCC4. When ectopically expressed in cells, the XIR-BRCT2 fragment can interact directly with XRCC4 to achieve strong dominant-negative effects, including radiosensitization associated with degradation of displaced endogenous LigIV, abated stable recruitment of XRCC4 and Cernunnos-XLF to damaged chromatin, and defective end-joining activity in cell extracts. In parallel, we demonstrated that Cernunnos-XLF was a DNA-. 2.
(8) PK substrate in vivo in response to DSB induction. Although deficiency in CernunnosXLF does not affect the recruitment of all the other NHEJ factors to damaged chromatin, its recruitment upon DSB induction essentially relies on DNA-PKcs, XRCC4 and LigIV. Finally, we determined that LigIV stabilizes the interaction between Cernunnos-XLF and XRCC4. Overall, our results provide new insights into XRCC4/LigIV/Cernunnos-XLF complex architecture that may enlighten our knowledge on the molecular mechanism of NHEJ and help in designing new adjuvants to anticancer chemo- or radiotherapy.. 3.
(9) •. Index. !. RÉSUMÉ .................................................................................................................. 1. !. ABSTRACT .............................................................................................................. 2. !. INDEX....................................................................................................................... 4. !. ABBREVIATIONS .................................................................................................... 7. !. LIST OF FIGURES AND TABLES......................................................................... 10. CHAPTER 1.. INTRODUCTION AND RESEARCH BACKGROUND......................... 12. 1.1. DNA damage and repair ............................................................................................................. 12 1.1.1. Endogenous DNA damage .................................................................................................. 12 1.1.2. Exogenous DNA damage..................................................................................................... 13 1.1.3. Repair of damaged bases .................................................................................................... 16 1.1.3.1. Base excision repair......................................................................................................... 17 1.1.3.2. Nucleotide excision repair................................................................................................ 19 1.1.3.3. Mismatch repair ............................................................................................................... 19 1.1.3.4. Single strand break repair................................................................................................ 20 1.1.4. Double-strand breaks repair................................................................................................. 21 1.1.4.1. Overview .......................................................................................................................... 21 1.1.4.2. DNA Damage Response.................................................................................................. 22 1.1.4.3. Homologous Recombination............................................................................................ 23 1.1.4.4. Non-Homologous End-Joining ......................................................................................... 25 1.1.4.5. Choice between HR and NHEJ........................................................................................ 25 1.1.4.6. Back-up pathway of NHEJ ............................................................................................... 26 1.2. Mechanism of Non-Homologous End-Joining ......................................................................... 28 1.2.1. Overview of NHEJ ................................................................................................................ 28 1.2.1.1. Involvement in V(D)J and class switch recombination..................................................... 28 1.2.1.2. Deficiency in NHEJ factors .............................................................................................. 30 1.2.1.3. Model of NHEJ repair pathway ........................................................................................ 32 1.2.2. Complex-1 of NHEJ pathway (DNA-PK) .............................................................................. 33 1.2.2.1. Ku heterodimer ................................................................................................................ 34 1.2.2.2. DNA-dependent protein kinase catalytic subunit (DNA-PKcs)......................................... 35 1.2.2.3. Accessory proteins and end-processing factors .............................................................. 38 1.2.3. Complex-2 of NHEJ pathway (ligation complex) .................................................................. 40 1.2.3.1. XRCC4............................................................................................................................. 40 1.2.3.1.a. Discovery of XRCC4.................................................................................................. 40 1.2.3.1.b. Structure and Domains.............................................................................................. 41 1.2.3.1.c. Celllular functions and modulation............................................................................. 45 1.2.3.2. DNA Ligase IV ................................................................................................................. 46 1.2.3.2.a. Overview of ATP-dependent DNA Ligases ............................................................... 46 1.2.3.2.b. Structure and BRCT domains.................................................................................... 49 1.2.3.2.c. DNA Ligase IV deficiency and mutations................................................................... 53 1.2.3.2.d. Interaction interface between XRCC4 and Ligase IV ................................................ 55. 4.
(10) 1.2.3.2.e. DNA Ligase IV stability .............................................................................................. 58 1.2.3.3. Cernunnos-XLF (XRCC4-like factor) ............................................................................... 59 1.2.3.3.a. An unknown NHEJ factor .......................................................................................... 59 1.2.3.3.b. Discovery of Cernunnos-XLF .................................................................................... 60 1.2.3.3.c. Deficiency and mutations........................................................................................... 61 1.2.3.3.d. Cernunnos-XLF and DSB repair................................................................................ 63 1.2.3.3.e. Cernunnos-XLF and ligation fidelity........................................................................... 64 1.2.3.3.f. Crystallographic structure of Cernunnos-XLF ............................................................ 65 1.2.3.3.g. Cernunnos-XLF interaction with other factors ........................................................... 67 1.3.. Conclusion and aim of the experimental study ....................................................................... 71. CHAPTER 2.. INTERACTION INTERFACE BETWEEN LIGIV AND XRCC4 ............ 75. !. Summary.......................................................................................................................................... 77. !. Introduction ..................................................................................................................................... 78. ! ! ! ! ! ! ! ! !. Results ............................................................................................................................................. 80 Structure of human XRCC4/LigIV complex .................................................................................. 80 Structure comparison of human XRCC4/LigIV ............................................................................. 81 XRCC4/LigIV interface.................................................................................................................. 83 Minimal XRCC4-interacting region in human cells ....................................................................... 84 Endogenous LigIV protein downregulation upon stable expression of LigIV fragments ............... 85 NHEJ protein assembly at DSBs in vivo upon stable expression of LigIV fragments. ................. 86 NHEJ activity upon stable overexpression of LigIV competing fragments.................................... 87 Cell survival after IR treatment upon overexpression of LigIV fragments ..................................... 88. ! ! ! !. Discussion ....................................................................................................................................... 88 Tandem BRCT domains of LigIV mediate different modes of protein interaction ......................... 88 The essential role of LigIV BRCT2 domain in XRCC4 interaction in vivo. ................................... 90 Phenotype of NHEJ-deficient LIG4 patients ................................................................................. 92 Implication for pharmacological perspects.................................................................................... 93. ! ! ! ! ! ! ! ! !. Experimental procedures ............................................................................................................... 95 XRCC4/Ligase IV crystallization, data collection and structure determination. ........................... 95 Construction of plasmid vectors expressing human LigIV fragments ........................................... 96 Cell lines, cell culture and transfection ......................................................................................... 97 Irradiation and survival assays ..................................................................................................... 97 RT-PCR ........................................................................................................................................ 98 Antibody and western blots........................................................................................................... 98 Recruitment assay ...................................................................................................................... 100 Immunoprecipitation ................................................................................................................... 100 In vitro NHEJ assay .................................................................................................................... 100. !. !. !. Figure legends............................................................................................................................... 107. !. Supplementary data...................................................................................................................... 121. ! ! ! !. Supplementary experimental procedures .................................................................................. 121 Immortalization of MEFs with SV40 T-antigen............................................................................ 121 Transient V(D)J recombination assay......................................................................................... 121 Supplementary figures and figure legends ................................................................................ 123. 5.
(11) CHAPTER 3.. INTERPLAY BETWEEN CER-XLF AND NHEJ FACTORS .............. 128. !. Abstract.......................................................................................................................................... 129. !. Introduction ................................................................................................................................... 129. ! ! ! ! ! ! !. Material and Methods ................................................................................................................... 130 Chemicals ................................................................................................................................... 130 Antibodies ................................................................................................................................... 130 Cell culture and cloning .............................................................................................................. 130 DNA-damaging treatments ......................................................................................................... 130 Biochemical fractionation and immunoblotting ........................................................................... 130 Co-immunoprecipitation assay. .................................................................................................. 131. !. Results ........................................................................................................................................... 131 ! Cer-XLF is phosphorylated by DNA-PKcs upon DSBs. .............................................................. 131 ! Cer-XLF is co-recruited with major NHEJ factors but is not necessary for their mobilization to damaged chromatin............................................................................................................................. 132 ! Cer-XLF mobilization to the damaged chromatin is dependent on XRCC4/LigIV. .................... 133. !. Discussions ................................................................................................................................... 134. !. Supplementary material and methods ........................................................................................ 136. !. Legends to supplementary figures ............................................................................................. 136. CHAPTER 4.. DISCUSSION AND PERSPECTIVES ................................................ 143. 4.1.. Interaction interface of LigIV/XRCC4 complex....................................................................... 143. 4.2.. A new approach for designing candidate LigIV inhibitors ................................................... 143. 4.3.. Minimal XRCC4-interacting region.......................................................................................... 145. 4.4.. Instability of full-length Ligase IV and Ligase IV fragments ................................................. 146. 4.5.. Integrated function of Cernunnos-XLF in NHEJ .................................................................... 146. 4.6.. Interaction network between Complex-2 factors ................................................................... 147. 4.7.. Architecture of Complex-2 and higher-order structure......................................................... 148. 4.8.. Models of NHEJ pathway ......................................................................................................... 150. !. REFERENCES ..................................................................................................... 153. 6.
(12) •. Abbreviations. A AID, Activation-Induced (Cytidine) Deaminase APE1, Apurinic/apyrimidinic endonuclease AT, Ataxia telangiectasia ATM, Ataxia-telangiectasia mutated gene ATLD, Ataxia telangiectasia-like disorder B Bax, Bcl-2-associated X protein BER, Base excision repair BLM, Bloom syndrome helicase B-NHEJ, back-up pathway of NHEJ BRCA1/2, breast cancer 1/2 genes BRCT, BRCA1 C-terminus domain BRCT1, first BRCT domain of LigIV BRCT2, second BRCT domain of LigIV C Caspase, cysteine proteases family Cer-XLF, Cernunnos-XLF CD, catalytic domain CHK 1/2, Checkpoint homolog 1/2 genes CHO, Chinese Hamster Ovary C-NHEJ, classical NHEJ Complex-1, DNA-PK, comprising Ku70/Ku80/DNA-PKcs Complex-2, ligation complex, comprising XRCC4/ LigIV /Cer-XLF CSR, class-switch recombination D DBD, DNA binding domain DNA, Deoxyribonucleic acid DNA-PKcs, DNA-dependent protein kinase catalytic subunit DNA-PK, DNA-dependent protein kinase or Complex-1 Dnl4p, yeast homolog of human LigIV D-NHEJ, DNA-PKcs dependent NHEJ DSB, DNA double-strand break DSBR, DNA double-strand break repair E EM, electron microscopy ES, embryonic stem cells EXO1, DNA exonuclease 1 F F2RL2, Coagulation factor II (thrombin) receptor-like 2 FEN1, Flap endonuclease 1 FRAP, fluorescence recovery after photobleaching. 7.
(13) G GFP, green fluorescent protein GG-NER, Global genome NER H H2AX, histone H2A variant hCAP-E, human condensin associate protein E HR, homologous recombination I Ig, immunoglobulin IR, ionizing radiation K Ku70, XRCC6, Ku autoantigen, 73-kDa Ku80, XRCC5, Ku autoantigen, 86-kDa L LigI, LigIII, LigIV, ATP-dependent DNA Ligase I, Ligase III, and Ligase IV Lif1p, yeast homolog of human XRCC4 LIG4 syndrome, human LigIV deficiency syndrome. Ligation complex, XRCC4/ LigIV/Cernunnos-XLF or Complex-2 LOH, loss of heterozygosity M MHEJ, microhomology mediated end-joining MMP-9, Matrix Metaloprotease 9 MMR, Mismatch excision repair MRE11, meiotic recombination 11 homolog MRN, Mre11/RAD50/NBS1 complex N NBS1, Nijmegen breakage syndrome gene 1 or Nibrin NCD, non-catalytic domain NER, Nucleotide excision repair NHEJ, Non-homologous end-joining Nijmegen breakage syndrome (NBS) NK, Natural killer cells NLS, nuclear localization signal O OBD, Oligo-binding domain P p53, tumor protein p53 PARP1, Poly (ADP-ribose) polymerase 1 Par-3, protease activated receptor 3, or F2RL2 PCNA, Proliferating cell nuclear antigen PI3K, Phosphoinositide 3-kinases family PNK, Polynucleotide kinase Pol X, Polymerase X family. 8.
(14) PPi, inorganic pyrophosphate R RAG1 and RAG2, recombination activating genes 1 and 2 RFC, Replication factor C RNA, Ribonucleic acid ROS, Reactive oxygen species or oxygen radicals RPA, Replication protein A RS-SCID, severe combined immunodeficiency with radio-sensitivity RSSs, recombination signal sequences S SCID, severe combined immunodeficiency SDSA, synthesis-dependent strand annealing SSA, single-strand annealing SSBR, DNA single-strand break repair SSB, DNA single-strand break ssDNA, single-stranded DNA SUMO, small ubiquitin-related modifier T TCR-NER, transcription-coupled repair of NER TCR, T-cell receptor TDP1, tyrosyl-DNA phosphodiesterase 1 TdT, terminal deoxynucleotidyltransferase Top1, Topoisomerase 1 U UV, Ultraviolet W WRN, Werner syndrome helicase/3'-exonuclease X XIR, XRCC4-interaction region of LigIV XLF, XRCC4-like factor XR-1, X-ray sensitive CHO cells 1 XRCC, X-Ray-sensitive-Cross-Complementing group Y YFP, yellow fluorescent protein Others 53BP1, tumor protein p53 binding protein 1 or TP53BP1 !H2AX, phosphorylated species of histone H2A variant X. 9.
(15) •. List of figures and tables. Chapter 1 Fig. 1. Potent DNA DSBs inducers. Fig. 2. Base excision repair pathway Fig. 3. Mismatch repair pathway Fig. 4. Causes of DSBs and the consequences Fig. 5. Homologous Recombination repair pathway Fig. 6. V(D)J recombination Fig. 7. Immunoglobulin (Ig) heavy chain class switching Fig. 8. General model of NHEJ pathway Fig. 9. Domains map of Ku70/Ku80 proteins Fig. 10. Crystallographic view of Ku heterodimer Fig. 11. Physical map of DNA-PKcs Fig. 12. Cryo-EM images and 3D reconstructions of DNA-PK/DNA Fig. 13. Possible mechanism for the control of the processing in NHEJ Fig. 14. Sequence alignment and secondary structure of XRCC4 homologs Fig. 15. Crystal structure and domains of XRCC4 Fig. 16. Molecular surface presentation of XRCC4 tetramer Fig. 17. Alignment of LigIV interaction and tetramerization region of XRCC4 Fig. 18. Summary of the activities affected by truncated forms of XRCC4 Fig. 19. Universal ATP-dependent DNA ligation mechanism Fig. 20. Domain structure of ATP-dependent DNA Ligases Fig. 21. Physical map of LigIV conserved domain and motifs Fig. 22. Relative positions conserved motifs and domains of LigI on DNA Fig. 23. Steps of ATP-dependent ligation Fig. 24. Position of BRCT domain in BRCT superfamily Fig. 25. Crystallographic structure of an XRCC1 BRCT domain Fig. 26. Positions and phenotypes of mutations found in LIG4 patients Fig. 27. Crystallographic structure of XIR-XRCC4 complex Fig. 28. Interaction interface of XIR-XRCC4 complex Fig. 29. Crystallographic structure of yeast DnL4p-Lif1p Fig. 30. Map of Cer-XLF domains and mutation positions Fig. 31. Crystallographic structure of Cer-XLF monomer Fig. 32. Crystallographic structure of Cer-XLF dimmer Fig. 33. Sequence aligment of different eukaryotic Cer-XLF homologs Fig. 34. Interaction domains between XRCC4 and Cer-XLF Fig. 35. Possible tripartite interaction network within NHEJ Complex-2 Fig. 36. Inhibitors that target DNA repair and damage response pathways. 16 18 20 22 24 29 30 33 34 35 36 36 40 42 43 43 44 44 47 48 49 50 51 52 53 54 56 56 57 62 66 66 67 68 69 72. Chapter 2 Table 1. Figure 1. Figure 2. Figure 3. Figure 4.. Crystallographic data and refinement stastics Structure of XRCC1-203 bound to DNA LigIV645-911 Structure alignments of XRCC4-LigIV structure Interface interactions of XRCC4-LigIV complex DNA LigIV constructs.. 10. 109 110 111 112 113.
(16) Figure 5. Interaction between XRCC4 and LigIV fragment in vivo Figure 6. Characterization of stably transfected MRC5 cells expressing LigIV fragments Figure 7. Consequence of LigIV fragment expression on NHEJ protein mobilization in vivo in response to DSBs Figure 8. Effect of LigIV fragment expression on DNA end-joining activity in vivo. Figure 9. Consequence of LigIV fragment expression on survival to IR Figure S1. Interaction between XRCC4 and LigIV fragment in vivo Figure S2. Structure comparison of BRCT domains from LigIV and BRCA1 Figure S3. Arg814 hydrogen bonding network Supplemental Table 1. Transient V(D)J recombination ssay Supplemental Table 2. Sequence of coding junctions from V(D)J recombination assay. 114 115116 117118 119 120 123 124 125 126 127. Chpater 3 Figure 1. Detection of Cer-XLF in extracts from human cells Figure 2. Effect of a defect in DNA-PK activity on the DSB-induced phosphorylation of Cer-XLF Figure 3. NHEJ proteins analysis after fractionation of untreated and calicheamicin-treated human cells. Figure 4. Effect of Cer-XLF defect on NHEJ proteins mobilization in response to DNA DSBs. Figure 5. Consequences of various NHEJ defects on Cer-XLF mobilization in response to DNA DSBs. Figure 6. Analysis of Cer-XLF and XRCC4/LigIV co-immunoprecipitation in extracts from LigIV deficient and proficient cells. Supplementary Figure 1. Comparison of recruitment of NHEJ and BER proteins in P2 fraction of MRC5cells after treatment with Cali. and MMS Supplementary Figure 2. Effect of Cer-XLF defect on NHEJ proteins mobilization in response to DSBs. Supplementary Figure 3. Consequences of various NHEJ defects on CerXLF mobilization in response to DSBs in HeLa clones. Supplementary Figure 4. Consequences of LigIV defect on Cer-XLF mobilization in response to DNA DSBs in MRC5 cells. Supplementary Figure 5. Immunoprecipitation controls.. 131 132 132 133 133 134 138. 139 140 141 142. Chapter 4 Fig. 37. Binding pocket of LigIV/XRCC4 interface Fig. 38. Relationships between Complex-2 factors Fig. 39. Filament model for assembly of XRCC4/LigIV/Cer-XLF complexes Fig. 40. Barrel-like model for assembly of XRCC4/LigIV/Cer-XLF complexes. Fig. 41. Possible models of NHEJ pathway.. 11. 144 148 149 150 152.
(17) Chapter 1. Introduction and research background. 1.1.. DNA damage and repair. Deoxyribonucleic acid (DNA) is the most fundamental element for all known live forms on earth. The genes, comprised by numerous, repetitive and complex segments of DNA, store the genetic information as well as command the normal development and functioning in all living organisms and most of the viruses. These genetical messages carried by DNA can be passed to RNA then translated to substantial working proteins, allowing the latter continue to drive all the components running like a machine and even constructing as a whole cell. So important the role it plays, DNA has to be kept stable for a long time to ensure the accuracy of information it contains. However, DNA in the cells is easily to be damaged by both endogenous and exogenous agents during the life cycle of cells. These damages might have immediate and long-term costs. If lesions occur at the strand of genes that are currently used by cells, they block the transcription for messenger RNA and impede the subsequent production of encoded proteins; the gene expression is instantly interfered by lesions. If an injured DNA template is duplicated during the replication cycle, this error will be possibly converted into mutations that change the genetic information permanently. Mutations can lead to protein malfunctioning, inherent defects (if carried by germ-line cells), and cancer. In this regard, the articulated network of genome must equip certain type of caretaking mechanisms for protecting the DNA from damages and consequently lethal outcomes. An elaborate reservoir of various corresponding DNA repair pathways has evolved that, in general, is capable to recognize virtually any type of DNA lesions, and to reverse them back to harmless counterpart. To this extent, a transient cell cycle arrest is needed to postpone DNA replication and create a time window for allowing repair machineries to eradicate lesions before being converted into permanent mutations or fetal chromosomal aberrations. If the damage is too severe, the final security mechanism, apoptosis, has to be utilized that makes damaged cells committing suicide and prevent the abnormal cells taking places to endanger the entire organism.. 1.1.1.. Endogenous DNA damage. DNA damages can be categorized to two major origins, endogenous way and exogenous way. The first kind of endogenous DNA damage is oxidative stress, generated by reactive oxygen species (ROS) and created during normal metabolic processes, that attacks DNA. Attacking at the sugar group will cause DNA fragmentation, base loss, and strand interruption. Attacking at the base will make more than 80 different products (Bjelland and Seeberg, 2003) such as thymine glycol (Demple and Linn, 1982) and 8-. 12.
(18) oxo-7,8-dihydroguanine (8-oxoG) (Kennedy et al., 1997). Both the strand-breaks generated directly by ROS and the aberrant bases generated in the attacking process by ROS can be recognized by lots of DNA repair mechanisms. Endogenous damages could also result from the spontaneous alterations in DNA base linking that makes DNA phosphate backbone hydrolyzed and chain broken (Friedberg et al., 2006). Also, deamination of cytosine, adenine, guanine, and 5methylcytosine, as well as depurination and depyrimidination occurs slowly at physiological condition could also provide modified bases as the targets for DNA repair. Besides that, mismatched DNA bases during replication is another kind of endogenous damage, in which the incorrect and nocomplementary DNA base is incorporated into newly synthesized daughter strand of DNA, or a DNA base has been skipped for synthesis or mistakenly inserted by DNA polymerase. Although the DNA polymerases have proofreading ability to ensure the fidelity, mistakes can still be made at a frequency of 1 out of 106 nucleotides (comparing to 1 out of 103 to 105 without proofreading) (Friedberg et al., 2006). Except chemical alteration, endogenous DNA damages can be also made programmedly by specialized cell mechanisms, particularlly in developing lymphocytes. For example, during the maturation steps of immunoglobulin and T-cell receptor genes in B and T lymphocytes, the genome segments harbor those genes will be excised by RAG1 and RAG2 (recombination activating genes) proteins at specific sites and religated by DNA repair mechanism. This step leads to recombination of immunoglobulin genes and is essential for providing diversity of antibody and T-cell receptor (Abbas and Lichtman, 2003). Same thing happens in the immunoglobulin class switching process, DNA breaks are generated at conserved motifs of immunoglobulingenes constant-region by a series of enzymes, including Activation-Induced (Cytidine) Deaminase (AID), uracil DNA glycosylase and apyrimidic/apurinic (AP)-endonucleases (Casali and Zan, 2004; Durandy, 2003) before being repaired. Class-switching recombination can help anibody changing its isotype. The mechanism of V(D)J recombination and class switching will be described latter in non-homologous endjoining section (1.2.1.1).. 1.1.2.. Exogenous DNA damage. There are numerous types of DNA damage caused by extra cellular agents, including ultraviolet (UV) radiation from the sun, ionizing radiation (IR) including x-rays and gamma ray from radioactive decay, nuclear fission, nuclear fusion or cosmic ray, naturally chemical and physical alteration like hydrolysis or thermal disruption, some toxins from food sources, and human-made mutagenic chemical compounds as those being applied for cancer therapy. The latter kind varies widely and covers alkylation agents, crosslinking agents, electrophilic reactants, oxidative agents, and radiomimetic strand-breaking agents.. 13.
(19) Ionizing radiation (IR) causes DNA damages in direct and indirect way (Breimer, 1990; Friedberg et al., 2006). The direct effect of of IR comes from the absorption of high energy by the surrounding layer of water-molecules bound to DNA. This can cause the ionizing of its bases and sugars to directly break down the DNA molecules. The indirect effect results from reactions of various free radicals generated by radiationionized cellular-water, including hydroxyl radical, hydrated electron, and H atom. These reactive oxygen species (ROS), especially hydroxyl radical, create multiple attacks on the DNA bases and the sugar-phosphate backbone. These attacks produce a lot of base-derived and sugar-derived products in DNA molecules, while they also produce protein-DNA crosslink in nucleoprotein (Dizdaroglu, 1992; Oleinick et al., 1987). Both DNA single strand and double strand breaks can be induced by IR (Roots and Okada, 1972; Sapora et al., 1991). Although by reports, the fraction of X-ray irradiation induced double strand breaks are about 5% of total lesions, whilst it is believed that the doublestranded breaks are the most lethal effects of IR due to the difficulty to be repaired (O'Driscoll and Jeggo, 2006; Tucker, 2008). Alkylating agents are electrophilic compounds that are able to attack nucleophilic center in organic macromolecules such as DNA by adding alkyl groups to negatively charged groups. They can be either monofunctional or bifunctional, and many of which are proven to be carcinogens (Lawley, 1989). They can cross-link guanine nucleobases in strands of DNA, which directly damages the DNA. Environmentally, alkylating agents include methyl chloride, streptozocin, and S-adenosylmethionine. Synthesized alkylating agents include: Methyl methanesulfonate, nitrogen mustards, melphalan, chlorambucil, cyclophosphamide, mitomycin C, dacarbazine, and procarbazine (Izbicka and Tolcher, 2004; Pukhalsky et al., 2006; Sedgwick et al., 2007; Yamada, 2004). By these synthesized chemical agents, DNA can be induced to form intrastrand crosslink or interstrand crosslink. Bifunctional alkylating agents as nitrogen mustards, sulfur mustards, cisplatin, mitomycins can crosslink DNA. If they attack the nucleophilic centers located on the opposite strand of DNA, the interstrand crosslinks are formed and the transcription as well as DNA replication are totally blocked due to DNA unable to uncoil and separate. This makes them useful chemotherapeutic agents, especially for quick replicating cells. When treat by these agents, even though cells may not die immediately, they also cannot grow. UV and ionizing radiations can also induce DNA crosslinks as minor products of irradiation. Crosslinks can also be formed between DNA and protein by aldehydes. A common and established application of this is to use formaldehyde to crosslink DNA with histone proteins in the chromatin immunoprecipitation assay (Perez-Romero and Imperiale, 2007; Toth and Biggin, 2000). Photoactivatable agents also generate another type of crosslinks. When compounds like psoralen and its derivatives are photoactivated after intercalating DNA, monofunctional or bifunctional adducts may form between pyrimidines and compounds. Bifunctional adducts can react from one pyrimidine to the one in the opposite strand thus crosslinking the two strands (Hearst et al., 1984). This intrastrand adduct cause DNA helical distortion, twisted, and even unwinding. Natural or synthetic enzymes and antibiotics also create DNA strand breaks. Topoisomerases are enzymes with both nuclease and Ligase activity. Type I of them. 14.
(20) can transiently nick one strand of DNA double helix allowing the other strand to rotate for reducing helical tension, then re-seal the nicked strand (Champoux, 2001). Type II of these enzymes act by cutting both strands of one DNA double helix simultaneously and then passing another unbroken strands of DNA double helix through this break before rejoining in order to reduce the DNA linking number (works in both direction to relax or twist) (Schoeffler and Berger, 2005). Topoisomerases are important due to the topological nature of DNA that requires these enzymes for unwinding DNA and proceeding replication and transcription (Wang, 2002). Inhibitors for topoisomerases stall the above mechanisms thus create DNA breaks; Topoisomerase I inhibitors like irinotecan, topotecan, camptothecin and lamellarin D cause DNA single strand breaks, while topoisomerase II inhibitors like amsacrine, etoposide, etoposide phosphate, teniposide and doxorubicin generate DNA double strand breaks. For example, etoposide trap a type II topoisomerase when covalently linked to a broken DNA end during DNA replication in the cell cycle, thereby creating protein-DNA crosslinks with impaired strand breaks. This makes these inhibitors so powerful and popular targets for cancer chemotherapy (Ferguson and Baguley, 1996). Bleomycin is a glycopeptide antibiotic, which was first identified from Streptomyces verticillus and proved having anti-cancer activity (Umezawa et al., 1966). It now refers to a structurally related compounds family and widely used for treatment of Hodgkin lymphoma, squamous cell carcinoma, testicular cancer, pleurodesis as well as plantar warts and so on. It works by inducing DNA strand breaks by chelating metal ions thus producing a pseudoenzyme that reacts with oxygen to produce superoxide and hydroxide free radicals that cleave DNA (Claussen and Long, 1999; Povirk, 1996). Neocarzinostatin is a macromolecular chromoprotein antibiotic secreted by Streptomyces macromomyceticus. It is shown to have anti-tumoral activity by its DNA damaging activity and related to macromomycin, actinoxanthin, kedarcidin and maduropeptin as the neocarzinostatin group of antibiotics. Its structure comprises two parts, a labile chromophore and a tightly, non-covalently bound apoprotein. The chromophore is very labile thus relied on the apoprotein to chaperon it to the target DNA. This makes it a very potent DNA-damaging agent (Kobayashi et al., 2006; Povirk, 1996) (Fig. 1). Calicheamicin is also an antibiotic, which was first derived from bacteria Micromonospora echinospora ssp. Calichensis (Maiese et al., 1989). This compound was then found to be an incrediblely potent cytotoxic agent by efficiently destroying DNA in tumor cells such as murine P388 leukemia and B16 melanoma in vivo (1,000 to 10,000 times more than other commercial drugs when treating on normal cells) (Zein et al., 1988). It now belongs to the enediyne group of antibiotics family which can release a non-diffusible 1,4-dehydrobenzene-diradical species that initiates oxidative strand scission by hydrogen abstraction on the deoxyribose ring of nearby DNA molecule to cause double strand breaks (Fig. 1). This site-specific DNA cleavage is triggered and activated only when the compound uniquely fits to the minor-groove of the DNA double helix (Zein et al., 1988). Comparing to irradiation causing less than 5% double strand breaks of total lesions, the calicheamicin can produce more than 30% double strand breaks of total lesions in vitro (Elmroth et al., 2003), therefore it is capable of working at very low concentration and believed to be a promising anti-cancer chemotherapy agent.. 15.
(21) The recent application of this compound, for avoiding attack on non-tumor cells, was conjugating it to anti-tumor monoclonal antibody. CD-33 is a cell marker expressed on most of (>80%) acute myelogenous leukemia (AML) cells surface but not normal hematopoietic cells, which makes it a perfect candidate for cancer targeting. The first commercial drug — MylotargTM in the new drug class, antibody-targeted chemotherapy agents, was therefore designed according to this idea, conjugating calicheamicin onto anti-CD33 monoclonal antibody to allow the DNA damaging efficacy being directed to tumor cells and bypass normal cells (Hamann et al., 2002; Hinman et al., 1993), and successful used in the treatment of AML.. Fig. 1. Potent DNA DSBs inducers. Scheme shows the chemical structure of (A). Neocarzinostatin and (B). Calicheamicin. The chromophoe of Neocazinostatin is labeled with diamond and the enediyne group of Calicheamicin is labeled with square. (C). The releasing of diradical intermediate from the functional group of two compounds that is capable of attacking nearby DNA. The oxygen radicals are labeled with starts.. 1.1.3.. Repair of damaged bases. The damaged or false-incorporated bases of DNA are removed mainly by excision repair pathways including base excision repair (BER), nucleotide excision repair (NER), and mismatch excision repair (MMR) (Hsieh and Yamane, 2008; Li, 2008; Saldivar et al., 2007; Shuck et al., 2008; Zharkov, 2008); whilst a very marginal fraction is repaired by. 16.
(22) direct damage reveral (photoreversion, alkyltransfer) (Lindahl, 1974). Generally speaking, BER is responsible for repairing those small base modifications that do not distort the DNA helix (uracil and 3-methyladenine), NER is in charge of repairing covalent DNA alterations that distort DNA helix (pyrimidine dimmers and benzpyrene adducts), and MMR recognizes and corrects single mispaired or mismatched normal DNA nucleotide involving several bases.. 1.1.3.1. Base excision repair Base excision repair (BER) is a multi-steps repair pathway that eliminates individually damaged bases including those products formed by hydrolysis, deamination, reative oxygen species, and monofunctional alkylating agents. It was identified by chance when searching for enzyme acting on deaminated cytosine (Lindahl, 1974). The first discovered enzyme, uracil DNA glycosylase, also the first of its kind, works by releasing the damaged bases which causes minor alteration in DNA structure, including alkylating adducts like 3-methyladenine, oxidative adducts such as thymine glucol and 8-oxoguanine, deamination of cytosine to uracil, and those errors in DNA replication including misincorporation of dUTP or 8-oxo-dUTP (Zharkov, 2008). The BER pathway starts from a damage-specific step acted by one of the DNA glycosylases and continues by damage-general steps performed by different proteins that correct DNA by inserting one or multiple nucleotides at damaged site in templatedirected manner. The removal of damaged bases is the only catalytic function of monofunctional DNA glycosylases, in which each enzyme has its own substrate preference (Krokan et al., 1997). The resulting abasic sites, also can spontaneously occur by hydrolysis, are cytotoxic and mutagenic and have to be further processed either by short-patch or long-patch pathway as describing later. The subsequent core BER reaction takes places by the apurinic/apyrimidinic endonuclease (APE-1, also known as Hap1, Apex, Ref1) (Mol et al., 2000; Xu et al., 1998), which generates strand incision at the abasic site. Poly (ADP-ribose) polymerase (PARP) then binds to it and is further activated by DNA strand breaks (Shall and de Murcia, 2000); also, the newly identified polynucleotide kinase (PNK) may involve in protection and trimming the DNA ends for later synthesis when BER is initiated from single strand breaks produced by IR (Bernstein et al., 2005; Whitehouse et al., 2001; Zharkov, 2008) (Fig. 2). In the short-patch BER pathway, DNA polymerase " is recruited upon direct interaction with APE1 to perform one-nucleotide gap-filling reaction (Bennett et al., 1997; Fortini et al., 1998). This polymerase " dependent BER pathway is dominant (Bennett et al., 1997; Nealon et al., 1996; Sobol et al., 1996), performing 75 ~ 90% of all BER in human cells (Sattler et al., 2003). After removing of 5’-terminal baseless sugar residue by lyase activity of polymerase ", the XRCC1-Ligase III complex then join to seal the remaining nick by presumably XRCC1 as the scaffold protein interacting with all above core factors and helping for protein turn over (Bennett et al., 1997; Cappelli et al., 1997; Nealon et al., 1996; Sobol et al., 1996; Tomkinson and Mackey, 1998; Zharkov, 2008) (Fig2).. 17.
(23) Fig. 2. Base excision repair pathway (Hoeijmakers, 2001). BER pathway starts from DNA glycosylases remove damage bases thus resulting abasic sites (I). Abasic sites can also arise from spontaneous hydrolysis (I, right panel). APE1 takes place to incise DNA strand (II). BER can also starts from X-ray induced SSB (upper right panel). PARP-XRCC1 binds to SSB and is activated (upper right panel). PNK comes to protect and trim the ends for repair synthesis (III). In mammals, short-patch BER dominates the remainder steps (lower left panel). DNA pol" performs a one-nucleotide gap-filling reaction (IV) and removes the 5’-terminus baseless sugar residue by its lyase activity (V). XRCC1-Ligase 3 complex continues to reseal the nick (VI). XRCC1 serves as scaffold protein that interacts with most of BER facotrs and may instruct for protein exchange. In minor subset, the long-patch BER pathway works by DNA pol#, pol$, PCNA perform repair synthesis (2–10 bases) (VII), followed by FEN1 remove the displaced DNA flap (VIII). DNA Ligase1 reseals the final nick (VIII).. The long-patch BER pathway repairs damaged bases by synthesizing 2-10 nucleotide stretching at the damaged site (Deterding et al., 2000; Gary et al., 1999). This synthesis performs by polymerase # and $ (Matsumoto et al., 1999; Pascucci et al., 1999), as well as polymerase " (Dianov et al., 1999; Klungland and Lindahl, 1997; Lindahl et al., 1997). Polymerase " mediated long-patch BER is not always required but is stimulated by proliferating cell nuclear antigen (PCNA) (Klungland and Lindahl, 1997). However, on the contrary, repair synthesis though polymerase # and $ depends on PCNA (Stucki et al., 1998). Since the polymerase " deficient cells were ever proved to. 18.
(24) be deficient in short-patch BER but proficient in PCNA-dependent long-patch BER, polymerase # and $ are believed to be the main polymerases responsible for long-patch BER (Fortini et al., 1998; Sobol et al., 1996). After repair synthesis, the displaced DNA flap is removed by flap endonuclease 1 (FEN1) (Klungland and Lindahl, 1997), and the nick can be resealed by DNA Ligase I upon the interaction to PCNA (Levin et al., 1997; Zharkov, 2008) (Fig2).. 1.1.3.2. Nucleotide excision repair In contrast to BER pathway that only cleaves damaged bases, nucleotide excision repair (NER) pathway excises and removes an oligonucleotide fragment containing the damaged bases. There are two sub-pathway of NER that deploy on distinct lesion types (Friedberg et al., 1995), in which the global genome NER (GG-NER) scrutinize throughout the whole genome for helix-distorting changes (Shuck et al., 2008), and the transcription-coupled repair (TCR) works on lesions which blocking the transcriptional elongation process (Tornaletti and Hanawalt, 1999).. 1.1.3.3. Mismatch repair Mismatch repair (MMR) corrects mismatched or mispaired (but normal, that is nondamaged) nucleotides generated by the fact that DNA polymerases and multiple-bases insertion/deletion loops generate sliding and differential errors when replicating repetitive sequences or during DNA recombination (Li, 2008). Defects in MMR pathway cause the microsatellite instability thus increasing genome mutation rate and the possibility for cancer (Jiricny, 1998; Shuck et al., 2008). In human MMR pathway, hMSH2 is in charge of the initial recognition for mismatched nucleotides during the postreplication MMR process (Fishel et al., 1994a; Fishel et al., 1994b; Fishel et al., 1994c). It further interacts with hMSH6 or hMSH3 to form heterodimers which performing redundant mismatch-binding activities regarding to the type of lesions (Acharya et al., 1996); in which hMSH2/6 (called hMutS%) complex is responsible for mismatches and single-base loops, while hMSH2/3 dimer (hMutS") detects insertion/deletion loops. Other heterodimeric complexes like hMLH1/hPMS2 (hMutL%) and hMLH1/hPMS1 (hMutL") are then recruited by the hMSH complexes and interact with replication factors (RFC) (Bronner et al., 1994; Li, 2008; Nicolaides et al., 1994; Papadopoulos et al., 1994). After strand discrimination, the excision step for removal of wrong (newly synthesized) strand past the mismatch and also the re-synthesis step for the excised tract are done by a panel of proteins including polymerase #/$, replication protein A (RPA), PCNA, (Hsieh and Yamane, 2008; Li, 2008), suggesting MMR components also interact functionally with NER, BER and recombination (Fig. 3).. 19.
(25) Fig. 3. Mismatch repair pathway (Hsieh and Yamane, 2008). Scheme shows the steps of eukaryotic MMR pathway in 3’-directed repair. MMR starts by MutS% (MSH2–MSH6) or MutS% (MSH2– MSH3, not shown) and MutLa (MLH1– PMS2) recognize a mismatched site and form a ternary complex. Their interactions within the complex and with DNA are modulated by ATP/ADP cofactors bound to MutS% and MutS% (red stars). PCNA may recruits MMR proteins to the adjacent replication fork through MSH6 and MSH3. PMS2 is stimulated by ATP, PCNA, and RFC (green arrow) and then performs endonuclease function to nick the DNA. It may establish strand discrimination and direct repair system to the newly synthesized strand. (MMR is bidirectional; 5’-directed repair is not shown) Excision by EXO1, and possibly other undefined exonucleases leads to the formation of an RPA-coated single-strand gap. Resynthesis is done by replicative pol# and ligation by Ligase I.. 1.1.3.4. Single strand break repair DNA Single strand breaks (SSBs) can be produced by a large variety of genotoxic agents either directly including IR and oxidative agents, or indirectly like alkylating agens. The latter usually relates to normal intermediates of excision repair. The repair of above SSBs is now termed as single strand break repair (SSBR). Generally, SSBR utilizes almost the same route as BER; therefore, some researchers tend to categorize SSBR as part of BER (Watson et al., 2004). Among different SSBs, the direct SSBs are repaired by short-patch BER pathway, whereas indirect SSBs are repaired by longpatch BER pathway as described in earlier section (Caldecott, 2001, 2003).. 20.
(26) Another example for indirect SSBs is generated by Topoisomerase 1 (Top1) during its normal catalytic cycle where it covalently attaches to the 3’-terminus of break before subsequent resealing (Wang, 2002). At this step, if the ligation activity of Top1 is inhibited due to encounter of other nearby lesions or some inhibitors, a so-called abortive top1 SSB occurs (Pouliot et al., 1999). To repair this, PARP and TDP1 (tyrosyl phosphodiesterase) (El-Khamisy et al., 2007; Takashima et al., 2002) are recruited to SSBs, and the 3’-top1 peptide is removed by TDP1 thus leaving a nick with 3’phosphoate and 5’-hydroxyl termini. PARP then recruits XRCC1-Ligase III% heterodimer and is replaced by this complex at breaks. Finally, PNK is next recruited to repair the 3’ and 5’ termini and let Ligase III% seals the remaining nick (Caldecott, 2003; El-Khamisy et al., 2007).. 1.1.4.. Double-strand breaks repair. 1.1.4.1. Overview Among all the DNA lesions in the cells, double-strand breaks (DSBs) are the most hazardous ones. When only one of the two strands of DNA double helix is damaged, no matter the kind of lesion and its origin, the second strand can still serve as a template. However, when both strands are affected, e.g. during intermediate steps of V(D)J and class-switch recombination (van Gent and van der Burg, 2007), or following exposure to ionizing radiations or radiomimetic compounds, or when replication fork encounters single-strand breaks (Lopes et al., 2001), intrastrand or interstrand crosslinks (Bessho, 2003), or stabilized topoisomerase-II cleavable complexes (Adachi et al., 2003), the cellular outcome is of most concern (van Gent et al., 2001). It is now broadly accepted that a single unrepaired DSB is lethal while a mis-repaired DSB can result in chromosome aberration, genomic rearrangement and possibly tumorigenesis (Khanna and Jackson, 2001; Mills et al., 2003; Pfeiffer et al., 2004; van Gent et al., 2001) (Fig.4). To cope with such cytotoxic lesions, cells have evolved two major pathways to deal with DSBs: the homologous recombination (HR) pathway that requires the presence of homologous DNA strand as a template, and the non-homologous endjoining (NHEJ) pathway that requires no or only little homology to restore DNA coninuity. The detailed mechanisms of these repair pathways will be described in next Sections. HR repair pathway works virtually by the same enzymatic machinery accountable for chromosomal crossover during meiosis. The DNA template mostly derives from a sister chromatid (i.e. only available in late S and G2 phases of the cell cycle) (San Filippo et al., 2008). When a homologous template is not available, the cells turn to utilize NHEJ to directly rejoin the broken ends (Weterings and Chen, 2008). The NHEJ pathway is a rapid event that works virtually immediately after damage induction. Its mechanism involves a DNA-dependent protein kinase (comprising a large catalytic subunit, DNA-PKcs, and a DNA-end-binding heterodimer Ku70/Ku80), DNA Ligase IV, XRCC4 and Cernunnos-XLF as core factors. It is also referred to as D-NHEJ according to its dependence upon DNA-PK or C-NEHJ standing for classical NHEJ pathway as opposed to alternative DNA-PK-independent pathways also referred to as backup. 21.
(27) NHEJ pathway (B-NHEJ) or microhomology-mediated end-joining (MHEJ) that can remove most of DSBs in the absence of NHEJ and HR factors, although with slower kinetics (Iliakis et al., 2004; Nussenzweig and Nussenzweig, 2007).. Fig. 4. Causes and consequences of DSBs (edited from van Gent et al., 2001). 1.1.4.2. DNA Damage Response According to a classical picture, following DNA damage infliction, the DNA damage response (DDR) consists in suspending cell cycle and, according to damage severity, to allow DNA repair machinery to proceed with the lesions or to trigger apoptosis (Bree et al., 2004; Harper and Elledge, 2007; van Gent et al., 2001; Zhou and Elledge, 2000) (Fig. 4). DNA damage response is triggered by autophosphorylation of the protein kinase dimer, Ataxia-telangiectasia mutated (ATM) at a.a. residues S1981 resulting in its subsequent monomerization (Bakkenist and Kastan, 2003). ATM is believed to be essential for the cellular response to DSBs. Upon DSB induction, ATM is rapidly activated and further phosphorylates downstream mediators or effectors (NBS1, BRCA1, CHK1, CHK2, p53, etc.), resulting in cell cycle arrests in the G1/S transition, S phase or G2/M transition (Abraham, 2001; Bakkenist and Kastan, 2003; Lukas et al.,. 22.
(28) 2004; Valerie and Povirk, 2003). Activation of ATM appears to occur with a distance from the DSB, and it is suggested that the changes in higher-order chromatin structure caused by DSBs are responsible for triggering ATM activation (Bakkenist and Kastan, 2003). However, ATM has also been shown to be recruited to lesion sites, at which it phosphorylates downstream substrates in response to DSBs (Lukas et al., 2003) by at least two possible mechanisms. In the first one, ATM recruitment relies on the MRN complex (described in the next section) (Falck et al., 2005; Lee and Paull, 2005), while the second one it is dependent on p53-binding protein-1 (53BP1), which binds to methylated histones that are exposed by chromatin-structure alterations in response to DSBs (DiTullio et al., 2002). ATM was also shown to phosphorylate DNA-PKcs, suggesting a role in the NHEJ process (Chen et al., 2007; Shrivastav et al., 2008). In addition to participate in cell cycle checkpoint and NHEJ repair, ATM also phosphorylates a variant of histone 2A, namely H2AX, upon DSB induction (Cowell et al., 2005; Stiff et al., 2004). H2AX amounts to 2-25% of the histone H2A total pool in mammalian cells and is randomly incorporated into nucleosomes. H2AX are rapidly phosphorylated (within a minute after DSB induction) in regions flanking a DSB and over several kilobases up to a megabase (Rogakou et al., 1998). This huge accumulation of phosphorylated H2AX proteins, designated as !H2AX, at DSB site can be easily detected as focus by immunofluorescence staining and visualized by microscopy. !H2AX foci reach a maximum in 15-30 minutes following irradiation and then decrease due to DSB repair, thus making it a useful and conventional DSB marker (Rogakou et al., 1998; Rothkamm and Lobrich, 2003). From a more mechanistic point of view, it is also suggested that !H2AX concentrates DNA repair proteins and prepares the chromatin for subsequent DNA repair events (Fernandez-Capetillo et al., 2004; Lowndes and Toh, 2005).. 1.1.4.3. Homologous Recombination Eukaryotic homologous recombination repair (HR), also known as templateassisted repair, homology directed repair (HDR) or recombinational repair (RR) (West, 2003), is triggered by the DNA damage response. As described in the previous section, upon DDR activation, ATM phosphorylates H2AX, which then binds several factors including MDC1, BRCA1 and NBS1, thereby allowing the recruitment of the whole MRN nuclease complex (Mre11/RAD50/NBS1) to DSB. ATM can further phosphorylate BRCA1 and NBS1 (Petrini, 2000), and BRCA1 may then regulate the downstream RecA-like RAD51 recombinase in damage response and assist the alignment to sister chromatid (Cousineau et al., 2005; Scully et al., 2004). In addition to BRCA1, BRCA2 may also participate in the process by regulating both the nuclear translocation and DNA-binding of of RAD51 (Davies et al., 2001). After recognizing DSBs and being activated, MRN may cooperate with other nucleases to perform 5’&3’ exonucleolytic resection and converts the DSB ends to 3’protruding ends (D'Amours and Jackson, 2002) (Fig. 5A). The resulting hundreds bases long single-stranded DNA (ssDNA) (Petrini, 2000) will be pre-bound by replication protein A (RPA), thus facilitating the assembly of a nucleoprotein filament that. 23.
(29) comprises RAD51 and RAD51 paralogs including XRCC2, XRCC3, RAD51B (or RAD51L1), RAD51C (RAD51L2) and RAD51D (RAD51L3) (San Filippo et al., 2008; Thacker, 1999). RAD52 and RAD54 proteins participate also in the reaction, perhaps with the help of the BLM and/or WRN helicases (Valerie and Povirk, 2003), in which they can stimulate the presynaptic complex formation as well as strand invasion (Shinohara and Ogawa, 1998; Sung, 1997). The tumor suppressor p53, known to interact with BRCA1, RAD51, BLM and WRN, is also likely to be present in this DNAprotein complex. The nucleoprotein filament is responsible for searching homologous sister chromatid throughout the genome and invading the intact duplex DNA thus forming a triple-stranded DNA structure called displacement loop or D-loop. Subsequently, the intact double-stranded DNA served as a template for DNA synthesis to recover intact genetic information (Valerie and Povirk, 2003). From this step, HR can proceed either by synthesis-dependent strand annealing (SDSA) or double-strand break repair (DSBR) (San Filippo et al., 2008) (Fig. 5A).. Fig. 5 Homologous Recombination repair pathway (edited from San Filippo et al., 2008; Valerie and Povirk, 2003). (A) Upon DNA damage response, activated ATM phosphorylates H2AX that serves both as a nucleation signal and amplification device for the binding of subsequent factors. The MRN complex cooperates with nucleases to resect the DNA and generate 3’-protruding ssDNA overhangs that are necessary for DNA pairing, strand invasion and strand exchange. BRCA2 is next attracted to the DSB. 24.
(30) by BRCA1 and facilitates the loading of RAD51 onto RPA-coated ssDNA with the help of RAD51 paralogs. RAD52 and RAD54 are also necessary for the presynaptic complex formation and subsequent strand invasion, perhaps with the help of the BLM and/or WRN helicases, and presumably p53. The resulting nucleoprotein filament searches homologous DNA (sister chromatid) and pairs with and invades the intact duplex DNA thus forming the D-loop. The intact copy of DNA is used as a template for DNA synthesis. HR completion can proceed either through a synthesis-dependent strand annealing (SDSA) mode(B) that produces noncross-over product, or a double-strand break repair (DSBR) mode (C) associated with Holliday junctions (HJ) formation. Holliday junctions can be resolved by non-crossover (black triangles) or crossover (gray triangles) associated events. All these reactions are finalized by gapfilling DNA synthesis and ligation.. In SDSA, the D-loop is unwound and one of the freed ssDNA anneals with complementary counterpart from the opposite side of DSBs end. This reaction is then completed by gap-filling synthesis and ligation (Fig. 5B). Only noncross-over products are formed in this subpathway. In DSBR, the second DNA end is captured to form an intermediate DNA structure with two Holliday junctions. These interweaved holidayjunctions are next processed by resolvases (Haber, 2000; Khanna and Jackson, 2001; van Gent et al., 2001) that probably consist of XRCC3/RAD51C complex (Brenneman et al., 2002; French et al., 2002; Liu et al., 2004), followed by gap-filling DNA synthesis and ligation. Holiday Junction resolution can result in crossover or gene conversion, depending on the reciprocal transfer, or not, of flanking genetic material, respectively (Fig. 5C).. 1.1.4.4. Non-Homologous End-Joining NHEJ directly reseals broken DNA without need of homology sequence. It is now categorized as the classical pathway (C-NHEJ) or the DNA-PK dependent pathway (DNHEJ), distinct from a backup pathway (B-NHEJ), possibly overlapping a recently builtup microhomology-based, DNA-PK independent end-joining pathway (MHEJ) (Kabotyanski et al., 1998; Nussenzweig and Nussenzweig, 2007; Verkaik et al., 2002; Weterings and Chen, 2008). To date, there are six core factors have been identified to be involved in C-NHEJ including Ku70, Ku80, DNA-PKcs, Ligase IV, XRCC4, Cernunnos-XLF, whilst the factors and mechanism contributing to B-NHEJ have not been fully characterized. The detailed mechanism and factors involved in C-NHEJ will be described in Section 1.2. and the concept of B-NHEJ is described in Section 1.1.4.6.. 1.1.4.5. Choice between HR and NHEJ NHEJ and HR pathways are both important and necessary for elimination of DSBs in all eukaryotes; however, little is known about how the choice between two pathways is made in cells. While HR is the major pathway in yeast, NHEJ seems to be the prevalent mechanism in higher vertebrates and mammalians (Critchlow and Jackson, 1998; Dudasova et al., 2004). The choice between pathways is mostly dependent on the cell-cycle machinery. As mentioned in previous paragraph, HR appears active when the sister chromatid is present during the late S and G2 phases. It is demonstrated that NHEJ predominates the DSBs repair during G0 and early S phases, while a minor. 25.
(31) activity in other phases has also been reported (Burma and Chen, 2004; Haber, 2000; Lee et al., 1997; Rothkamm et al., 2003; Saintigny et al., 2001; Shrivastav et al., 2008; Takata et al., 1998; Valerie and Povirk, 2003). On the other hand, although HR and NHEJ appear to collaborate to maintain genome stability, it has also been suggested that a competition mechanism seems to determine the use of pathway for DSBs repair in cells. Since both HR and NHEJ have been shown to have the ability to contact the same lesion site (Richardson and Jasin, 2000), the binding of RAD51 and RAD52 HR factors to broken ends possibly competes with the NHEJ DNA binding protein Ku heterodimer (Baumann and West, 1998a, b; Haber, 2000). Supporting this possibility, inhibition of NHEJ activity significantly promotes the HR mediated repair (Allen et al., 2002; Delacote et al., 2002; Fukushima et al., 2001; Pierce et al., 2001). Notably, this promotion of HR only takes place when the early stage protein of NHEJ, DNA-PK (comprising DNA-PKcs and Ku complex), is knocked-out but not latter stage proteins like XRCC4, suggesting the competition between the HR and NHEJ occurs during the lesion binding and recognition steps (Pierce et al., 2001). On the other hand, spontaneous and DSB-induced HR activities are also decreased upon the kinase inactivation of DNA-PKcs by chemical inhibitors (apparently the DNA-PKcs still occupies the end although un-functional) but not in the absence of DNA-PKcs protein, supporting an “interactive competition” model between HR and NHEJ (Allen et al., 2003).. 1.1.4.6. Back-up pathway of NHEJ Aside from HR and NHEJ (or C-NHEJ), a distinct repair pathway was recently brought up, namely B-NHEJ (Lin et al., 1999; Nussenzweig and Nussenzweig, 2007). The most significant feature of this alternative NHEJ pathway is that the end-joining step needs to be mediated by DNA sequence microhomolgy of 1-8 bp (Kabotyanski et al., 1998; Verkaik et al., 2002); therefore, it is also termed as microhomology-mediated NHEJ (MHEJ) (Liang et al., 2005; Liang et al., 2008). The signature of this pathway usually consists in nucleotide deletions at the joined site flanked by microhomoloy sequences (Feldmann et al., 2000; Kabotyanski et al., 1998). The existense of this distinct, alternative, and perhaps optional NEHJ pathway was heavily debated as it may be simply a variant of the single-strand annealing mechanism with low joining efficiency (SSA, homology dependent but RAD51 independent pathway, predominantly described in yeast or Xenopus, but uncertain in higher eukaryotes (Decottignies, 2007; Gottlich et al., 1998; Paques and Haber, 1999)) or may result from the incomplete activity of NHEJ factors (Lieber et al., 2004; Paques and Haber, 1999; Verkaik et al., 2002). However more and more evidences tend to support it as a real alternative pathway (for review, see Nussenzweig and Nussenzweig, 2007). At present time, the mechanism and components involved in B-NHEJ await to be precisely deciphered. It has been shown that efficient end-joining can occur in the absence of the core NHEJ factors including DNA-PKcs, Ku, LigIV, and XRCC4 (Audebert et al., 2004; Kabotyanski et al., 1998; Liang et al., 2008; Verkaik et al., 2002; Wang et al., 2003; Wang et al., 2001b), supporting that B-NHEJ is clearly distinct from. 26.
(32) C-NHEJ. Furthermore, when measuring the removal of IR induced DSBs, it was shown in vivo that the operating half-time by NHEJ is about 10-30 min or at least less than 3 hrs. (Iliakis et al., 2004; Jenner et al., 1993); whereas a different repair kinetics of repair after inhibition of known NHEJ factors shown a longer operating half-time of about 2-10 hrs (Iliakis et al., 2004; Wang et al., 2001b), suggesting B-NHEJ is a slower and different route than the conventional NHEJ mechanism. This slow and C-NHEJ independent B-NHEJ pathway still keeps a DSB rejoining ability in mutants lacking several genes from the RAD52 epistasis group, indicating it is also independent of HR (Wang et al., 2001a). Furthermore, C-NEHJ appears to suppress B-NHEJ by the DNAends binding ability of DNA-PK (Perrault et al., 2004; Wang et al., 2003), suggesting a competition or regulation between the two pathways. Finally, a breakthrough in finding B-NHEJ components recently linked double strand break repair to DNA base excision repair. It was demonstrated that the short-patch BER factors PARP1/XRCC1/Ligase III perform the synapsis formation and end-joining activity (Audebert et al., 2004; Wang et al., 2005), and both Ligase III and Ligase I (long-patch BER factors) but not C-NHEJ factor Ligase IV are required for microhomology-mediated DSBs end-joining (Liang et al., 2008). Both Ligase III and Ligase IV can efficiently ligate broken ends at very low protein level (Windhofer et al., 2007). Additionally, histone H1 promotes the ligation capacity of both Ligases, while more pronounced in the case of Ligase III and accompanied with PARP1 stimulation (Rosidi et al., 2008). In agreement with above finding, PARP1 has been shown to compete with Ku on the damaged site for the repair of DSBs (Wang et al., 2006). Thus, C-NHEJ appears fast and efficient, and deals with DSBs with relative high fidelity, whereas B-NHEJ appears to be slower and more error-prone. In this regard, CNHEJ may be an evolutionary optimized pathway. Indeed, DNA-PKcs, which is absent in yeast (Daley et al., 2005), may have evolved to facilitate and speed up the reaction at least by stabilizing the synapsis formation for the subsequent ligation step. On the opposite, B-NHEJ could be an evolutionary older mechanism that rejoins lesions by borrowing excision repair factors (Iliakis et al., 2004).. 27.
(33) 1.2.. Mechanism of Non-Homologous End-Joining. 1.2.1.. Overview of NHEJ. C-NHEJ (in subsequent Sections of this work, we will term C-NHEJ as “NEHJ” pathway for more convenience) is virtually the most important mechanism upon all the DNA repair systems that prevent us from immediate and continuous DNA damages in our daily life. Its defect gives rise to immunodeficiency, genome instability, senescence, apoptosis, and tumorigenesis (Jeggo, 1998b; Lieber, 2008; Nussenzweig and Nussenzweig, 2007; Pastwa and Blasiak, 2003; Poplawski and Blasiak, 2005; van Gent and van der Burg, 2007; Weterings and Chen, 2008). NHEJ is an efficient, rapid, and conceptually simple pathway. Briefly, the basic molecular mechanism involves proteins that detect, capture and bridge the two termini of broken DNA, and proteins that clean and finally ligate broken ends. So far, core factors identified as involved in the NHEJ pathway include a PI3 kinase holoeenzyme that consists of the Ku70/Ku80 heterodimer and the DNA-dependent protein-kinase catalytic-subunit (DNA-PKcs), the three tightly associated proteins XRCC4, CernunnosXLF (XRCC4 like factor), and DNA Ligase IV, and additionally, several accessory factors, including non exclusively Artemis, DNA polymerases, TdT, APE1, and PNK (Weterings and Chen, 2008). The repair model of NHEJ pathway will be detailed in Section 1.2.1.3. Ionizing-irradiated cell models have shown that NHEJ efficiently removes the majority of induced DNA breaks within a short period of time In addition to the repair of exogenously produced DSB, the NEHJ pathway is also required for the resealing of breaks produced during endogenous cellular processes like V(D)J recombination and class-switch recombination.. 1.2.1.1. Involvement in V(D)J and class switch recombination To adapt the immune response to the variety of antigens, the developing B- and Tlymphocytes undergo genomic rearrangement by randomly selecting and assembling V(D)J gene segments which encode the variable region of immunoglobulin and T cell receptor proteins (Fig. 6A). During this V(D)J rearrangement, the RAG1 and RAG2 (Recombination Activating Gene) proteins recognize and excise at specific sites, namely Recombination Signal Sequences (RSSs), flanking the sets of V, D, and J segments and generate covalently sealed DNA hairpins (McBlane et al., 1995; Ramsden et al., 1996; Schlissel, 2002) (Fig. 6B). Before the ligation step, these hairpins must be opened and processed further by the NHEJ factors; namely, DNA-PKcs regulates Artemis that exhibits both exonuclease and endonuclease activity for blunting 5’ ends and trimming 3’ overhangs (Ma et al., 2002) (Fig. 6B). In support of this, deficiency or mutations in Artemis or DNA-PKcs result in severe combined immunodeficiency (SCID) phenotype with no T or B cells (Moshous et al., 2001; Noordzij et al., 2003; Schuler et al., 1986). After end processing of hairpins, the resulting DNA ends are re-joined efficiently by NHEJ system (Soulas-Sprauel et al.,. 28.
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