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Structural insight into RAD18-RAD6 complex

Dans le document The DART-Europe E-theses Portal (Page 101-105)

5. RAD18: a key regulator of cell response to DNA damage

5.1. Structural insight into RAD18-RAD6 complex

The human RAD18 cDNA was cloned by screening a human placenta cDNA library using a human EST clone that encodes a peptide with homology to the N-terminus of S.

cerevisiae RAD18 and N. crassa UVS2 protein (Tateishi et al., 2000). The isolated hRAD18 gene encodes a protein of 495-amino acids and an estimated molecular weight of 56 kDa displaying an altered electrophoretic mobility since the protein is detected as an 80 kDa protein by western blot. Human RAD18 shares 20% sequence identity and 42% similarity with yeast RAD18, and both of them have a conserved ring-finger motif and a zinc-finger motif in the N-terminal domain (Figure 5. 1) (Tateishi et al., 2000). Using Fluorescence In Situ Hybridization (FISH) method and PCR mapping by using a radiation hybrid panel, the RAD18 gene has been mapped to a single locus on chromosome 3p24-25 (Tateishi et al., 2000). RAD18 localizes mainly in the nucleus.

RAD18 is a multidomain E3 ubiquitin ligase (Figure 5. 1) that has been reported to dimerize in vivo and in vitro (Masuda et al., 2012; Miyase et al., 2005; Notenboom et al., 2007;

Ulrich and Jentsch, 2000). Dimerization occurs via an N-terminal region (Hedglin and Benkovic, 2015). Genetic data have previously shown RAD18 dimerization in yeast (Ulrich and Jentsch, 2000) and human cells (Miyase et al., 2005). In yeast, the RAD18-RAD18 interaction was mapped to the region comprising residues 83-248 that contains the Zinc Finger (ZnF) domain. Mutation in the human RAD18 ZnF domain (C207F) disrupts its dimerization.

Therefore, the ZnF domain was suggested to be the dimerization domain (Miyase et al., 2005).

More recent in vitro data do not support this conclusion since regions containing the ZnF domain (199–366) or isolated ZnF do not dimerize (Notenboom et al., 2007). Probably other domains and/or post-translational modifications are necessary for this dimerization. Consistent

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with this possibility, it has been shown that RAD18 ubiquitination favours the dimer formation and strengthens the interaction between two RAD18 molecules to form a RAD18•Ub–RAD18 (Zeman et al., 2014).The RAD18C207F mutant has been shown to ubiquitinate PCNA in vivo and in vitro following UV damage (Miyase et al., 2005; Notenboom et al., 2007), and RAD18-null cells complemented with RAD18C207F acquire resistance to DNA damaging agents, including UV, mitomycin C, MMS, and cisplatin (Miyase et al., 2005; Tateishi et al., 2000).

Interestingly, cells expressing RAD18C207F are sensitive to ionizing radiation, at least in G1-phase (Watanabe et al., 2009). This could be explained by the fact that the ZnF domain of RAD18 is required for its binding to ubiquitinated proteins at double-strand break sites (Huang et al., 2009; Notenboom et al., 2007). In addition, this mutant in the RAD18 ZnF domain is not ubiquitinated in cells (Miyase et al., 2005), but capable of auto-ubiquitination in vitro (Notenboom et al., 2007). The function of the ZnF remains unclear, in addition to its role in RAD18 dimerization, other reports suggest its possible contribution in DNA binding, , ubiquitin binding, and controlling the cellular location of RAD18 ((Bailly et al., 1994; Jones et al., 1988;

Miyase et al., 2005; Tateishi et al., 2000; Ulrich and Jentsch, 2000).

The RAD18 protein binds to the human homologs of the yeast RAD6 protein (UBE2A and UBE2B) through a conserved RING finger motif and a RAD6-Binding domain (R6B). The RAD6-binding domain is located at the C-terminus of RAD18 and interacts with the noncovalent ubiquitin interaction site on RAD6 (Bailly et al., 1997a; Ulrich and Jentsch, 2000).

RAD18D340–395 deletion mutant loses its interaction with RAD6 in vitro, although localization to damage is not affected (Watanabe et al., 2004). At its N-terminal part, RAD18 contains a Ring domain, common in E3ubiquitin ligases, which also helps RAD6 binding, independently of the R6B domain (Tateishi et al., 2000). Mutations in this region increase the sensitivity to DNA-damaging agents. Gel-filtrations experiments coupled to multi-angle static light-scattering show that RAD18 forms with RAD6 is a dimer of heterodimers where two RAD18 molecules are bound together through their zinc finger domain , and each of them is bound to RAD6 through the RING finger and RAD6 binding domains (Notenboom et al., 2007).

In addition, RAD18 also contains other domains that bindPCNA, RPA, or DNA (the SAP domain). RAD18 does not contain a classical PIP box, however, the N-terminal region of RAD18 contain a PCNA-binding motif that acts independently of the other domains in this region (Notenboom et al., 2007). Moreover, the exact binding site of RAD18 on PCNA remains unknown. Since overexpression of proteins characterised by high affinity to PCNA front side,

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such as p15PAF and p21, does not impair PCNA monoubiquitination (Soria et al., 2006; Toledo et al., 2013), it was suggested that RAD18 may not require access to the front side of PCNA for monoubiquitination. RAD18 binds also to ssDNA at stalled replication forks through its SAP domain, following DNA damage (Bailly et al., 1994; Nakajima et al., 2006; Notenboom et al., 2007). In the actual model, both the SAP and RPA-binding domains participate to the recruitment of RAD18 to RPA-coated ssDNA generated at uncoupled replication forks (Davies et al., 2008; Huttner and Ulrich, 2008; Notenboom et al., 2007).

RAD18 also contains ATPase domain which function is currently unknown. Disruption of this domain does not affect the DNA binding of RAD18 or the RAD6/RAD18 complex and is dispensable for the RAD18-RAD6 interaction (Bailly et al., 1997a; Bailly et al., 1997b; Jones et al., 1988). Note that, except the zing finger domain (Rizzo et al., 2014), the ring finger domain (Huang et al., 2011), and the RAD6 binding domain (Hibbert et al., 2011), the crystal structure of full length RAD18 is not yet resolved.

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Figure 5. 1Schematic representation of the RAD18 protein interactions (Notenboom et al., 2007)

The RAD18 ZnF binds to ubiquitin, while the SAP domain is a DNA binding domain. RAD6 interacts with the Ring domain (catalytic domain) and between residues 340 and 395 towards the C-terminus (RAD6 BD). Pol η binding domain is located between residues 401 and 445 and the PCNA-interacting region is contained within residues 16–366 (shaded in brown). Lysine residues K161, K261, K309 and K318 represents the four sites of RAD18 autoubiquitination, which are conserved between mouse and human RAD18.

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Dans le document The DART-Europe E-theses Portal (Page 101-105)