1.1 178
ENDOGENOUS RNase-SENSITIVE DNA POLYMERASE COMPLEX FROM RAT TISSUES:
CHARACTERIZATION OF THE REACTION AND THE PRODUCTS, AND OF THE DNA POLYMERASES RELEASED BY RNase-TREATMENT
A Thesis Submitted to the Faculty of Graduate Studies of Memorial University of Newfoundland
in Partial Fulfillment of the Requirenents for the Degree of Doctor of Philosophy
in the
Department of Biochemistry
By
Francesco Moranelli
May, 1977
To Suzanne
TABLE OF CONTENTS
PAGE ABSTRACT • • • • •
ACKNOWLEDCEHE.''TS • LIST OF FIGURES LIST OF TABLES • LIST OFSOiEMES LIST OF ABBREVIATIONS GLOSSARY•• • • •
LITERAnJRE REVIEW I. Introduction.
(1)
(H1) (iv) (viH) (a)
(xi) (dH)
II. (A) Classificat.ion of Replicative DNA Polymerases (B) React.ionMechanism of Replicat i veDNA Polymerases • III. Characte ris t.icsof Various Replicative DNA Polymerases • 11
(A) DNA Polymerase(J • • • • • • • • •
(a) Basic Distinguishing Features (b ) Purif ication .
(c) Properties • • (1) TelllPlate Specificit.y (11) Divalent cation Requiremen t (ii i) Effect.of MonovalentCations (iv) pHOptima • . • • (v) IsoelectricPo i n t s (vi) Size • • • . • . • (d ) Subcell ularLocal iza t.ion• (e) Inhibition Studies •. •.
11 11 11 13 13 14 15
"
11 IS 21 22
PAGE
(f) BiologicalPunct ion . 24
(B) DNA Polymerase
s
27(a) Basic Distinguishing Features . 27
(b) Purification 27
(c) Properties 2'
(1) Template Specificity . 2'
(11) Divalent Cation Requirement 2'
(iii) Effect of Monovalent Cations 30
(dv) pH Optima 30
(v) Isoelectric Point 30
(vi) Size 31
(d) Subcellular Localization 31
(e) InhibitionStudies 32
(f) Biological Function 32
(C) DNA Polymerase y 35
(a) Basic Distinguishing Features 35
(b) Purification 36
(c) Properties 36
(1) Template Specificity . 36
(11) Divalent Cation Requirement 39
(11i) Effect of MonovalentCations 39
(iv) pH Optima 40
(v) lsoelectric Point 40
(vi) Size 40
(d) Subcellular Localization 40
PAGE
(e) Inhibition Studies 40
(f) Biological Function 40
(0) Mitochondrial DNA Polymerase 41
(a) Basic Distinguishing Features 41
(b) Purification 42
(0) Properties 42
(i) Template Specificity 42
(11) Divalent Cation Requirement 44
(11i) Effect of Monovalent Cations 44
(iv) pH Optima 45
(v) Isoelectric Point 45
(vi) Size 45
(d) Submitochondrial Localh.ation 45
(e) Inhibition Studies 46
(f) Biological Function 46
(E) RNA-Directed and RNase-Sensit.ive Endogenously-
Templated DNA Polymerases 47
(a) Introduction. 47
(b) Reverse Transcript.ion with Bact.erial Systems 51 (i) Exogenously-Templat.ed Reaction Catalyzed
byE.ootc 51
(11) Endogenous RNase-Sensit.ive DNA
Polymerase 53
(0) Involvement. of RNA in DNA Synthesis in
Eukaryotic Cells 54
(i) RNA as a Primer in DNA Synthesis 54 (11) RNA as a Template in DNA Synthesis . 54 (d) Properties of the RNA-Direct.ed DNA Polymerase 55
PAGE (e) Nature of the Reaction Products of the RNA-
Directed DNA Pokyaerasea .• • • • . • • 58 (f) Biological Function of RNA-DirectedDNA
Polymerase • • • • • • • • 59
IV. Te rmi n a l Deoxynucleotidyl Transferase •• 62
V. Serological Analysis of DNA Polymerases • 64
PURPOSE AND APPROACH TO THIS STUDY. • • • • . • • • • • • • • • 67
MATERIALS AND METHODS I. Materials
(A) Animals
(B) Bf.ochemdea'l.e , Chemicals and Enzymes (C) Buffers and Solutions
II. Methods
(A) Preparation of Tissue Homogenates
6.
6.
71
73 (B) Preparation of the Endogenous Ly-Pemp l at es RS-DF
Activity from Rat Thymus. • • • • • • • •• • • 73 (C) Preparation of gxogenouaLy-Teepfa r ed Activities
Derived from the RS-DP CompLexfrom Rat Thymus. 74
(D) Isoelectric Focusing• .• • .• • . • 74
(E) Electrophoresis in Glycerol Gradients 76
(F) Sil1conizationof cf.assvare • • • • • 78
(G) Isolation of the ReactionProducts of the
Endogenously-Templated DNA Polymerase 78
(8) Buoyant Density Analysis • • • • • • • • • 80 (a) CsS0
4Density Gradient Centrifugation. 80 (b) Guanidinium.Cl-CsCl Density Gradient
Centrifugation • •• • •. • • • • . • 81
(c) Sucrose Density Gradient Centrifugation . (1) Sedimentation Velocity Analysis
(J) DNA Polymerase Assay • (K) Prot.einDetermination
RESULTS
PAGE 81 81 82 8'
1. Detectionand Part:ia1 Purificat ionoft.he Endogenously Templated Ribonuclease-Sensit.iveDNA Polymeraseof Rat Thymus • • • •• • • • • • • •• • •• • • • • • • • 85 (A) DNA Polymerase Activit iesin a Crude Ext.ractof
Rat Thymus . • • • • • •• • • •• • • •• • • • 85 (B) Partial Purification of Rat Thymus DNA Pol}'1Df!rase 87 11. Properties of the Endogenous RNase-Sensitive DNA PolYUlerase
(A) Requiremen ts. • • • •• • • • •• . • • •• • • . 91 (B) HetcropolymericNatureof the Nucleic Acid Region
Transcribed by the Endogenous RNase-Sensit.iveDNA Polymerase. • • • • • • • • • • • • • •. • • • • 91 (C) Preliminary Evidence in favor of a Template Funct.ion
for the RNA in the Endogenously-TemplatedR."l'ase- SensitiveDNA Polymerase • . • • • •• •• • • • • 93 (D) Propertiesof the Endogenous RNase-SensitiveDNA
Polymerase• • •• • • • • 95
(a) CatalyticProperties • • • • • • 95
(t) Time-Courseof the Reaction 95
(ii) Activi tyVersusEnzyme Concentrat.ion
Curves • • • 98
(iii) pH Optilla.la• • • •• • • • • 101 (tv) Divalentcation Requirements 10 4 (v) Effect of Potassium Chloride • 104 (b) InhibitionStudies • • • • • • • • 104
PAGE (i) Effec t of ActinomycinD and Distamyc i n
A on the Endogenously-Templated Activi ty. 10 4 (ii) Effectof N-ethylmaleirnideon the RS-OP. 110 (ii i ) Effect of Heparin . . . .. . . .• • . 110 (r v) Effec tof Polyamineson the RS-DPand
DNA Polymerase a • . •. . . 110 (c ) Physical Properti es of the Endogenous Activity . 113
(i) Effectof TritonX-IOO on th eEn d o ge n ou s RS-DPActivi ty . . . . .• . . . .. . . . 113 (ii) Buoyant Densityofth e Endogenous RS-DP. 115 (iii) Mol e c u larWe i gh tof theEndogenous
RS-DP . • • . • . • •. • •• • • . 118 (iv) Isoelectric Point of the Endogenous
RS-DP. . • . • • • .. • • .• . . . 118 III. Purifica tionof th e Endogenous RNase-SensitiveDNA
Polymerase .. . . • • .. 121
(A) Reasons for Further Purification 121
(B) General Observations . .. . . . 121
(C) Purification of DNA Po.l yme r a s e s from Rat Thymus . 123 (0) Purificationof the RS-DPfrom Rat Liver 132 IV. Analysis of the RS-DP Productof Rat Liver . . 140
(A) Effectof Nucleases and Alkali on the Product
of the Endogenously-Templated DNA Polymerase. 140 (B) Buoyant Density Analysis of the Products.. . 142 (C) Size of the RS-OP Product afterVarious Treatments. 147 V. Isolationof the DNA Polymerase Activities Associated
with the RS-DP Complex . • • • • • • . . . 154 (A) Effect of Nuc Je as e Treatment on the Release of ORA-
Di reeted DNA Pc Lymer a s e s frOlll the tndogenously-
Templated DNA Polymerase ComplexfromRat Thymus . . 155
PACE (B) Effect of Basic Proteins on the Endogenously-
TetDPiated DNA Polymerase Complex . 160
(C) Effect of RNase Digestion Products on the Endo-
genously-TeDlplated DNA Polymerase Complex 162 (D) Effect of RNase-Treatment on the Endogenous
Activity in the Presence of Protease Inhibitors 163 (8) Effect ofR"'~a8eon More Extensively Purified
Endogenously-Templated RS-DP Preparations 165 VI. Properties of the DNA Polyulerases Released by RNase-
Treatment of the Endogenous Activity. 169
(A) Requirements 169
(B) Catalytic Properties 17l
(a) Time-Course for the Peaks II and III
Activities 171
(6) Activity Versus Enzyme Concentrat.ion Curve
for Peak II 17l
(0) pHOptimum . 174
(d) Divalent Cation Optima 174
(a) Effect of Potassita Chloride 178
(f) Template Specificity. 182
(1) Effect of Actinomycin D on the Exo- genously RNA-Templated Activity with
the Peak III EnzyuJe 184
(g) "Ka's" for Native and "Activated" DNA
Templates Cor Peaks II and III 188
(CJ Inhibition Studies 188
(a) Effect of N-ethylmaleimide 188
(6) Effect of Heparin 192
(oj Effect of Ethidium Bromide . 192
(d) Effect of Organic Solvents 192
(a) Effect of PolY8Illines 196
PAGE (D) Physical Propertiesof th e DNAPolymerase sDerived
fro. the RS-DPCOlllplex . 20 0
(a ) Mol e c ul ar Weight • • • • •• • • • • •• • • 200 (b ) Ls oeLectrfc Poin tsoft.hePeak III Activ it y . 20 3
DISCUSSIO N. . 20 7
Evidence SuggestingR."'A-Direc tedDNASynthesisin Matlllll8lian
Cells . . • . . . •• . • 207
(A) Indirect Evidence 208
(B) Direct Evidence . 210
(C) Comparison of th e Endogenously-TemplatedRS-DPActivity froto RatThytllUSto thatfrom Oncogenic RNA Viruses.. 214
(a) PhysicalProperties. • 214
(b) Biochemica l Pro perties 216
II EvidenceSuggestiv eof Two UniqueDNAPo l ymer a s e sAssocfeted ...ith the Endogenously-Templated RS-DP Complex. • . • .• • • 218 III Relationship of PeakI Iand IIIActivitiesto otherEukaryotic
DNA Polymerases. • • • •• • • • • •• •. . • . .• • . . . 221 IV Speculations on the Possible Functions of the RNA-Directeod DNA
Polymeorast! in NOI'lllaI Ma-.alianCeLf s , .. . . .. . . . • • 223
APPEXDlXI
Relationship betve e n Density and Refrac tiveIndex of Guanidiniuro.CI-CsCI Solutions.
REFERENCES • .• . . . •.. • • . . .
PrBLICATIONS ARlSING FROM THlS ""ORK
227
229
251
(1) ABSTRACT
Anendogenously-templated DNA poj.yeer-ase activityfrolllratth yurns and liver has been partially purified and characterized,and the product of the reactionanalyzed. The enzyme frombo th sources wa s shownto be sensitiveto pretreatment wit hRNases [h en c ei tisref e rr e dto as a R."'ase-sensi tiveDNApolymerase(RS-DP) ] . The molecularweight of the RS-DP complex, estimated from Sepharose 68 gelfiltrat ion ,is280,000 daltons.
The R."1A associated with the RS-DP is probably single-stranded (and therefore functions as a template) since t.he activity remained sensitive to gxese-e ree teeet;un d er conditions In which only single-strandedRNA Is dfge sted , The putative RNA templateis heteropolymericin nature, since all four nucleotideswereinco rpo rated intothe DNA producttoa similar extent (also indicating that the enayee is not simplya terminal transferase). The ene yee is probably not of viral origin. as the activity was not stimulated by non-ionicdetergents and also had ai buoyant density (1.05 gtcm3)
in a sucrose gradient vhf ch differed greatly from that reponed for the type-C viralactivity (1.22-1.24 gtcm3). tn- hibition studies wit.h act.inomycin D and distamycin A.however.revealed a similarit.y to t.he viral RNA-directed D:-lA polymerase (RD-DP).supporting the notion that the RNA in the RS-DP complexfunctions as a template.
Additional supporting evidence for a template function for theR.~Aderives from buoyant density analysis of the product of the rat liver RS-DP actIvf ry ,
The RS-DP activity differs from DSA-directed DSApoIy-ae r ase s in its preference forHn2+to !'Ig2+as the divalent cofactor. furthertllOre. the enz yee isDot inhibited by "'-ethyhc.aleimide and also responds dif-
(11)
ferently to heparin and polyamines than does the rat thymus DNA poly- .erase a.
Two lower molecular weight DNA polymerases 00,000 and 30-40.000 daltons. respectively denoted as Peaks II and III). were derived from the RS-DP complex by extensive RNase-treatment. These activities are probably not proteolytic fragments of a higher molecular weight DNA polyuterase. since RNase-treataaent in the presence of the protease inhibitors Trasyloland phenylmethylsulfonyl fluoride alsore s u l t e din the release ofthe s e activit.ies.
The PeakII and III activitiesdifferfrom each other in a number of t.heir properties. and furthermore,diffe r from other eukaryotic DNA polymerases described in the literature. indicating that the enzymes are dist.inct. and probably unique DNA polymerases.
(iii)
ACKNOWLEDGEMENTS
The autho r wis h e s to expresshis gratitude to the membersofhis Supervisory CollllDit tee ,Drs.P.E.Penner (Chaillllan),K.M.Keough,and C.-Y.Hew,for their helpfu l disc ussionsand suggestions,and especially for the i r critic a lcommentsin thewriting of thi s disse r ta tion.
The autho r is alsograt eful toMrs. MasumaRa h mi n tul a for herhelp in drawing some of the figures andto Mrs.Donna Osborne for herexcell- ent workintyp i n gth is dissertation.
Aspecial th a n k you is due to mywif e, Suzanne, for her encou rage - ment an dmora lsu ppo rtat ti me of need,an dfor he r assis ta nce in dra w- in g most of the figures.
Research support fr01llthe National and Medical Research Councils of Canada throughgrants toDr. P.E.Penner ,and fellowship support frOlll MemorialUnive rs ityof Newfoundlan d,arealsoacknowledged.
(tv)
LIST OF FIGURES
Rat Thymus DNA Polymerase Activities In the 39.000 x g Supernatant • •• • • . •. • • • • • • 86 Profile on Rat Thymus DNA Polymerases on a
Sepharose 6B Column • • • • • • •• . .• • 90 Time-Courseof the Endogenous RS-DFActivity 97 Sigmoidal Relat ionshipBetween Activity and Con- centration ofRS-DPComplex .• • • • • •• • • • 99 Inhibition ofRS-DPActivity at High Concentrations ofRS-DPComplex • • . . . • • • •• • • • • • 10 0 Activity VersusRS-DFConcentration Curves In
Siliconized and Non-SiliconizedTubes. • 102 pH Optimum of Endogenous RS-DPActivity• • • • 103 The Effect of Divalent Cations on the Endogenous RS-DPActivity • • • • • • • • • • • •• • • • 105 Effect of Potassium Chloride on the EndogenouB
RS-DF Activity • • •• • • • • • • • • • • • • 10 6 10
11
12
13
15
16
17
Effect of Actinomycin D 00 the Endogenous RS-DP Activity • . • • • • • • • • • • • . . • . . . Effect of Distamycio A on t.he Endogenous RS-DP and DNA Polymeraseaof Rat. Thymus • . . • • Effect of N-ethylmaleimide on the Endogenous RS-DPand DNA Polymeraseaof Rat Thymus •• Effect of Heparin on t.he Endogenous RS-DPand DNA Polymerase a of Rat Thymus • • • • • • • Effect of Polyamines on the Endogenous RS-DPand DNA Polymerase a of Rat Thymus • • •• • •• • Effect of TritonX-I00 on the Endogenous RS-DP Of Rat Thymus • • • •• • • • • . .. • . •• • Buoyant DensityAnalysisof the Endogenous RS-DP Act.ivity • • • • . • . • • • • • • • • • • • • • Molecular Weight Determination of the Endogenous RS-DPon a Sepharcae 6B Column • • • • . • • • •
108
109
III
112
114
116
117
119
18
19
20
21
22
23
24
25
zs
27
28
29
30
(v)
Isoelect ricFocusingof the Endogenous RS-DP
Ac ti v it y . • • • • •. . • . • •. • . . . .. 120 Gl y c e r ol Gradient Elec trophoresisPro fil e of
Rat Thymus DNA Polymerasesfromth e 39,000x
g Superna t an t • • • • • . • • •• • • • . 13 0 Glyc erol Gradien tElec t rophores isofthe
Endogenous RS-DPofFractionIV of th e Rat
Thymus Preparation . • . . • .. . • . . . 131 Act.ivityVersus Enzyme Concentration Curve for
theEndogenous RS-DPAc t i v it y from Fraction
IV8ofRat Liver . . . • . . . . • . . • . • 137 ActivityVersus Enzyme Concentration Curveof
Fraction V of the Endogenous RS-DPActivity
fr om RatLiver . • • • • • • •• • • •• • •• 13 8 Effectof Actinomycin Dand DistamycinA on the Endogenous RS-DP Activityfrom Rat Liver .• . 139 Effectof S1Nuclease Treatment on the Product Synthesize din the Pre se n c e of ActinomycinD,
BeforeandAfterHeat Denaturation . . • • • . lli3 Buoyant Density Analysis of the Product Synthesized in the Presence of ActinomycinD.on (A) Cs 2S0 4 and (B) CsCI-Guanidinium-ClGradients . . • • • 144 BuoyantDensity Analysisof the Product Synthe- sized inthe Presence of ActinomycinD,After (A) Heat Denaturation, (B) Alkali Treatment. (C) RNase A and Tl and (D) Nuclease S1
Treatment s . . . • . • • • •• • • • •• • • • 146 BuoyantDensity Analysis ofth e Product Synthe- sized in the Absence of Actinomycin D. Before
(A) and After (B) Al1ulli-Treatment •. • • • • 148 Sedimentation. VelocityAnalysisof the Product
Synt.be s Lzed in thePresence of ActinomycinD;
(A) Control. (B) After Heat-Treatment and (C)
After Alkali-Treatment .• • . • • • . • •. . ll.9 Sedimentation Velocity Analysis of the Product
Synthesized inthe Absence of Actinomycin D,
(A) Before and (B) After Alkali -Treatment •• 151 Elution Profile of Rat Thymus D:\A Pc Lytaer a s e s on a Sephadex G200 Column After Treatmentc-it.h
Various xccf eeses . • .• . . • . • • • • • • •. 15 6
FIGURE 31
32
33
34
3'
36
37
38
40 41
42
43 44
45
46
(vi)
Profile of the DNA-Directed DNA Polymerases on a Sephadex G200 Column After RNase A and Tl Treatment • •. • • . • • • • • • • • • • • Profile of RD-DP and DD-DPActivities on a Sephadex G200 Column After RNase A and Tl Treatment ofth e Endogenous Activity • • • Effect of Oxidized RNase A. Histones and Lacto- peroxidase on the Profile of the DNA Polymerases from RatThymus • . . • • • • • • • • • • • • • • DNA Polymerase Profile on Sephadex G200 of Native DD-DP After RNase-Treatment of the Endo- genously-Templated Activity in the Presence of (A) Tnsylol and (B) Phenylmethylsulfonyl Fluoride • • • • • • • • • • • • • • • . • • • Effect of Nucleases on Endogenously-Templated Activit.ies of Rat Liver Exposed to 400 mM KCl • Tae Course of the Peak 11 (A) and Peak III (B) Act.ivities Derived frOl:ll the RS-DP Complex • • • Activit.y Versus Enzyme Concentration Curves for Peaks II andIII •• • • • • • • • • • • • . . • Inhibition of the Peak 111 Activity at. High Levels of Enzyme • • • • • • • • • • • • • • • The Effect of pH on the Peak 11 Enz)'1lle • The Effect of pH on the Peak III Enzyme.
The Effect of Divalent Cations on the Peak 11 Enzyme • • • • • • • • • • • • • . . • • • • • The Effect of Divalent Cations on the Peak III Enzyme • • • • • • • • • • • • • • • • • • • • The Effect of KCI on Peak II and III Activities.
Dependence of Relative Efficiency of "Activated"
to xet tve DNA for Peak lIon Enzyme Concentration.
Apparent Km's for Native and "Act.ivated" DNA as Templates for the Peak II Enzyme • • • • • • • • Apparent KuI's for Kative and "Activated" DNA as Templat.es [or the Peak III Enzyme • • • • • • • •
157
159
161
16'
167
172
173
175 176 177
179
180 181
18'
189
190
~ 47
48
49
50
51
52
53
54
55
5.
57
(vii)
Effectof l'oi-ethylmaleimi deon (A) Peak.IIand (B) Peak III Enzymes• • . • • • • • • .• • • Effec t of Heparin on(A) PeakIIand (B) Peak III Enzymes • • • • • • • • • • • • • •• • • Effect of EthidiUllo Bromideonth e Peak II and III Enzymes • •• • • • • . • . • • • • • •. . . Effectof Polyamines on thePe a k. II and Peak III Enzymes • • •• •• • • • • • • .• • • . Effe c tofI m."IPut res c ineontheAct i v it y Versus Enzyme ConcentrationCu rvefor Pe a k sIIand II I Act ivi ties•• • • • • • • • • • • • • • • • • • Molecu la r Weight Est111l8. tionofPe ak IIand III Activi tieson a SephadexG200 Col umn• • • • • . Molecula rWeightEsti1ll8.t ionof thePeak II and IIIAct ivit ieson a Sepharose 6B ColUIllD ' . MolecularWeight Estimationof the Peak. II Act.ivi ty(A) Beforeand (B) After RNase- Treatmenton a Sepharose6B CoIUllln• • • • • Molecula rlo'eight Estilllationofthe Peak III Enzyme on a Sephadex G75 Column in the Presence of I H Salt " . • • • • • • • • •• • • • • Isoelect r icFocusing Analysisof the Peak. III Enzyme • • •• • • . •• • • • • • • • • • • Relations hipBetweenDensityand Refractive Ind ex of GuanidiniullI.CI -CsCI Solutions• . •
191
193
194
197
199
201
202
204
205
206
228
(viii)
LISt OF TABLES
Cos;arlson of Prope rtiesof DNAPolymerase 1,II and IIIofE.coli. . .• . .. •. • II Classification of ReplicativeDNA Polymerases III
IV
VI VII VIII
IX
XI
XII XIII
XIV
XV
XVI
XVII XVIII
Propertiesof DNA Polymerase a •• • • • • • • Conversion of High MolecularWeight DNA Pol)'lDf!rases to Lower Molecula rWeight Species Inhibitorsof DNA Polymerase0.•
Propertiesof DNA PolymeraseS • Inhibitors of DNA Polymerasel! • Propert.ies of DNA Polymerasey • TemplateSpecificity of DNA Polymerase Y. Propertie s of Mitochondria l DNAPol ymera s e . Criteria Used in Assigning a Template Fun ct i on toRNAinDNASynthesis • • • •• • • • • • • • Reaction Mixtureforth e DNA Polymerase Assays.
Partial Purification of Rat Thymus DNA Polymerases • • • • • • • • • • • • •• Requirellleotsof the RNase-SensitiveDNA Polymerase .• • • • • • • • •• • •. • Incorporationof 3H-Labelled Deoxynucleoside Honophosphates into DNA by the Endogenously- TemplatedRNase-Sensit ive DNAPol yme r a s e • • Sensitivity ofEndogenously-Temp la ted DNA PolymeraseAct ivityto RNase A and T
l inthe Presence of 200 m!1 NaCl . • • • • • •. • • Purification of Rat Thymus DNA Polymerases• • R.>,lase-Sensitivityof the Rat Thymus Endogenously- T_plated ActivityDuringth e Course of Purification
12
19 23 28 33 37 38 43
56 83
88
92
94
96 126
128
XlX
xx
xxr
xxII
XXIII
XXIV
xxv
:0....·1
XXVII
XXVIII
(ix)
Relative Specific Activity ofRat Thymus DNA PolYtllerases at Various Stages of Purification Purification ofthe Endodgenous RNase- Sensitive DNA PolymerasefrolllRatLiver RNase-Sensitivity of the Endogenously-Templated DNA Polymerase Activity from Rat Liver at Various Stages of Purificat.ion Effect of Nucleases and Alkali on the KS-DF Product
Analysis of the Sh.es of Products after Various Treatlllef\ts
Requirements of the DNA"Pol)"tlIerase a and of the Peak II and III Activities Derived from t.he RS-DF
Activity of Rat Thymus . • •
Templat.e Specificity ofRat Thymus DNA Polymerase e and the Peak III Activity Derived from the RS-DP Complex
Effect of Actinolllycin D on t.he Peak III Activity with DNA and RNA Templates
Effect of Organic Solvents on the Peak II and III Enz)'1Des
129
135
136
I ' l
152
170
183
186
187
195
(xl
LISTOF SCHEMES
Template-Primer Specificityof Replicative DNA Polymerases • • • • • • • • • • • • • • • • II Requirementsof Terminal De ox ynu c l e o t i d yl
Transferase •• • • • • • • • • • • •• • • III ReactionMechanism Proposed for DNA Polymerases •
IV ProvirusHypothesis• • • • . • . . . .• • • • • 49 Modes of Ribosomal Gene Amplifications(A) Semi- conservative Replication Model (B) Hodelof
Tacchini-Valentiniand Cripps. • • 60
VI VII
VIII
IX
Apparatus Used forEkectrc fccusdng • •• • Apparatus for Electropho resisin Glycero l Gradients. • • • . • • . . . .• • • • Flow -Cha rt for the Procedureused in Isolating th e Product of the RS-DP Reaction . Flo....-chart for the Purificationof Rat Thymus DNA Polymerases •• • . • • • •• • . . . . •
75
77
79
'24 Flow-Chartfor the Purification of the RNase
Sensitive DNA Polymerase from Rat Liver . . . 133 XI
XII XIII
Possible Hodes of Associationof the Endogenously Templated DNA Polymerasewith Nucleic Acids • Hypot.hetical Structures for the RS-DPProduct Central Dogma of Molecular Biology • • • • .
209 211-213 224
ADD-DP
>JoN araCTP B5A
CHT-GEN pCMS cpa CTAB dATP
dCTP DD-DP DEAE-cellulose dGTP DMSO DNA (rONA) DNase dsDNA DTT dTTP
EDTA EtOH LP NaPPi
(xi) LIST OF ABBREVIATIONS
"Activated" DNA""Directed DNA Polymerase Avian myeloblastosis virus
1-6-D-arabinofuranosylcytosine 5'-triphosphate Bovine serum albumin
Chymoc ryps Inogen p-chloromercur1benzoa t e counts per minute
cetyltrimethylammonium bromide 2'-de o x y r i bo s y l a de n i n e - S '-t.riphosphate 2' -deoxyribosylcyt id ine-S' -t riphosphate DNA-Directed DNA polymerase Diethylaminoethyl-cellulose 2' -deoxyribosylguanine-S'-t riphosphate dimethylsulfoxide
deoxyribonucleic acids (ribosomal) deoxyribonuclease
double-stranded DNA dithiothre1tol
2'-deoxyribosy1t.h)'lllidine-5'-triphosphat.e ethylenediamine tetraacetic acid ethanol
lactoperoxidase Sodium pyrophosphate
(xii)
NDD-DP NativeDNA-direc tedDNA Polymerase
NEM N-ethylmaleimide
OVA ovalbumin
PEG polyethyleneglyco l
PHA phytohaemagglutinin
pI isoelectric point
pmoles picomoles 00-12
moles ) pOHHgBZ p-hydroxymercuribenzoate PMSF phenylmethylsulfonyl flu o ri d e
RD-DP RNA-dir ec t e dDNA polymeras e(reversetranscriptase) RNA (rRNA,yRNA, ribonucleicacid(ribosomal,yeast,tobacco mosaic TMV-R....A. Q6RNA virus, phage Q6, haemoglobin message)
Hb-mRNA)
RNase ri b onu cl e a s e
rpm revolut ions per minute
RS-DP RNase-sensitiveDNA polymeras e
RSV Rous sarcomavirus
2-SH-EtOH 2-mercaptoethanol SDS sodium dode cy I sulfate Sp , Act. specific activit)'
TCA trichloroacetic acid
TdT te rtm.eaLdeoxynucleotidyl transferase Tris trfs (hydroxymet hy1) amino-me thane
(xiii)
GLOSSARY
In order to prevent any confusion that may arise frOfl. some of the terminology usedinthis dissertation. the following terms will be used as defined below:
Template: The nucleic acid strand dictating to the polymerases the sequence in which the deoxynucleotides will be incorporated.
Primer: The strand providing the 3'OH group essential for the initiation of DN... synthesis by ON'" polymerases.
...ctivated .. ON...: ON... partially digested with pancreatic DNase 1 for the purpose of increasing the number of 3'OHinitiation sites.
"Gapped" DNA: Exonuclease Ill-treated "activated" ON'" resulting in a stretch of single-stranded DNA.
RNA-directed DNA Polymerase: This term \o1ill be used ...hen the function of the RN'" is to act as a template.
R."IA-dependentDS'" Polymerase: This term will be used to indicate that the role ofR.~'"in the reaction is to act as a primer in thein- itiation of the reaction.
RNase-Sensitive DNA Polymerase (RS-DP) : Refers to the endogenously- templatedDNA polymerase that is sensitive to RNase-treatment.
- 1 -
LITERAnJRE REVIEW
1. lnt.roduction
DNA polymerases (DNA nucleotidyltransferase,EC 2.7 .7.7) .ubi- quitously distr ibutedin living organisms.are enzymes catalyzing the synthesis of DNA when suppliedwi t h the appropriate substrates. In order for an enzymatic reaction t.o occur,a DNA polymerase must be pro- vided with a template-prilller nucleic acid (DNA or RNA or combinations of).unless it is already associatedwi th an endogenous template-primer.
In addition itmustbe supp lied withthefour deoxynucleoside triphos- phst.'!1i and a diva len tcationsuchas magnesium or eanganeae , The sto i - chiometry, as well as some of th e template-prime rcombinationsused in t.he catalysis of DNA synthesis by suchenz)'lIIes. are depicted in Schemes ::: .1odII.
Onthe basis of template-primerorprimer requirement by DSA poly- mer a s e s (ccepare Schemes I and II), one can classify such enzymes into
twobasic categories (i) those that. cat.aIyze areplicative-t':Jp6reaction (Scheme I) and (11) those that catalyze a termi~ladditionreaction (SchelDe II) . The two classes are distinguishab leon the basis of whether they~or
ss
~require the directionof a DSA orRNAtemp Late withre- gard towh i ch deoxynucleosidetriphosphate willnext. be condensed int.h e polymerizat ionstep. In th e1'€pZiaative-typereaction , the sequence in which thenu c leo t i d e s are incorporated inthe newly synthesized DNAis det.e refned by th e DNAor RNA template, vhe eeas intheterminal addition reaction no template isre q u i r e d. andthe synthesized sequence 1s dete r-, mined byth e nucleoside triphosphate eade availabletoth e enzymes.The repZicative-ty~DSA po l yee r a ses may be further subdividedac-
>w
~
- 2-
- 3 -
REOUIREMENTS OF TE RM i N AL DEO XY N Il C I E ffi lD yt TRANSFERASE
"1'1"1"YOH + [ p-P Pl"oHl ~ 111"1"m1 '~-'
+[ P", L
T T
T T l
AI
n T T T T A A A A SCHEME IIcording to their template-primer specificities as well as on the basis of other distinguishingfeatures to be discussed in the next section.
II. (A) Classification of Replicative DNA Polymerases.
The D~ApolymerasefromE.col-i:initiallydiscoveredby Kornberg (1) ...as t.hought to functionin the semi-conservat.ive replicat.ionof DNA. The possible existence of mul tipleDNA pc l.yme re.s ea in p roke ryot.ea was not.
suspectedhowever until theteote rtc n by De Lucia and Cairns (2) of a mutant ofE.coli,lackingDNA polymeraseIbut stillcapable of normal DNA replication. Shortly after the discovery of Cairn's mutantre po r t s r-oncemIng DNA polymerase II (3), DNA polymeraseII I (4), and a complex form of DNA polymerase III(d e si gn a t e d as DNA polymeraseIII*) (5)ap- pea red in thelit e r a t u re . For the purpose of comparison,TableI out- lines some of the distingUishing featuresof DNA polymerasesI, IIand III ofE.coli.
The existence of multipleDNA polymerases (7-9) in eukaryotes was evident atan earlier stage of investigation thanin the prokaryotic system and was attributed to the higher complexity of eukaryotic organisms.
Within the past half decade,the compilationofdata concerning var ious eukaryotic DNA pc.Lymer-aae s has increasedto such an extent ,and the nomen- clature system has varied so euch that a tremendous amount of co~fusion
TABLE 11• Comparison of Properties of DNA Polymera&e I,II and III of E.coli
Propertlea DNA Polymeras e
1. .!..!. i l l
Molecula r Weigh t 109,000 120, 000 160,000
Prefer redTemplate "Ac t i va t e d" DNA2 "Ga ppe d"DNA3 "Ga pped"DNA
No. of Molecules/Cell 400 100 10
Associa tedExonuc1easea 51~3' 31~5' 3'--+5'
3'~5 '
Sensitivity to Sulfhydryl No 'res Y.,
Reagents
DeNovoDNA Syntheais te e No No
Inhibitionby Pol.I Ye , No No
Ant iserum
Inhibitionby araCTP 4 No Yes No
EHectof 10%Eton inhibition inhibition atilllulation
Structural Genes potA po! B dnaE
Abridged from Kornberg and Kornberg (6).
"Ac t i va t ed " DNA - DNA partially digested with pancreaticdeoxyribonuclease I,for the purpose of Inc reaa Ing the numb e r of3'OHinitiationsitea.
"Ga pped"DNA- exonuclease III-treated"a cti va t ed" DNA re s ul t i n g in stretchea (Le.,gaps) of single-strsnded regions in the DNA.
araCTP- l-B- D-Ara b i no ( u r a no s y l c yt osin e 5'-triphosphate
- 5 -
has evolved as to which "replicative"D~Apolymerase is actuallybeing referred tobydifferent inves tigato rs . Inan attemptto eliminate this confusion , a number of sc i e n t.i s t s have propose d auniform. nomencla t ure for identify i n geukaryot i cDNApofymerese s(10-12). Thenomen clat ur e systemisba sed on theuse of Greek let t ers in designatingthe DNA polymera8es inth e historical orde rin which theywe r e discovered.
analogous to the approach taken inth e naming of bacterial DNA polymer - ases (10).
TableIIoutlines five 1Il8in type sof replicative DNA polymerasesto be discussed in thisrevrev8Swellas some of their distingu ishing features. In addi t ion tothe s e fivetyp e s ,there arealsovira l -ind uce d D:\A polymerases whichwill not be considered here [see ve rs sbacb (10) for a brief review]. The table indicatesthat the classification isbased on a cceb Inetfcu of prope rtiessuch as size,tempLate specifici ty ,intra- cellularlocalization , and sensitivit yto sulfhydryl blockingreagents .
Previous atterup ts to cla s s if y DNApolymeras es wereba sed ei t he ron template specifici ty (14 ) . subcellula r lo c al h: a ti on (15),orsize (16).
Categorization according to telllpIate specificity alonehad to be abandoned however, since enzyme preparation frOlllvarious laboratories differinth e degree of contalllination by nucleases, whichwouldalte rthe t.e e p Iar e s and thus the enzymes' apparent specificity. Similarily , classification ac- cording to subcellu la r loc a l i z a t i onalon e is not satisfactory ,sincethe recovery of activity in various subcellularfractionsisin f l ue n c e d by the IsolatIonprocedure used in preparing the subcellularcoeponents (17).
The lIlitochondrialDSApol~raseIs the onlyeuka ryotfe DSA polymerase classified with certainty on the basisof its subcellular localization, sinee drastic conditions are nc rmaL'ly required to extract thispolymeras e
TABLE Ill. Classification o( ReplicAtive DNA PolymerBBeB
!
:L Mit. RD_Dp23.35 6.1-6.35 8·95 Type 13 70.000 Type II 170,000 Type III 110,000
C,N Hit. OncogenicRNA vi ruses
a
Properties DNA Polymerase
Subcel lula r Loc ali za tion4 Sedimentation 6-85
Coefficient or MolecularWeight
Inh i b i t io n by th iolblocki ng reagent s Nuclease Act i v ity Template
Specifici ty:
"a ct" DNA
rJ\
'dTl 5 din' r A1 0 heteropolymer i cRNA
Modified from Weissbach (l0) and Holmes And JohnBton (12).
AbbreviationBused: RD-DP--RNA-directedDNA polymerase,C -- cytop laslllic , N --nuclea r.Hit. -- mitochondrial.
According to Wu and Gallo (13).
Subcellula r localizat io nof the enayece,when Bubcellula rcomponen ts are pre pa r e dinbuHe re daqueous media.
- 7 -
from chat organelle (18). Classification on size alone also has its drawbacks, since the enzymes are well known for their ability to ag- gregate as well as to possibly associat.e with other proteins under certain conditions [e.g.,(19) also see secr toos ce Size below].
The RNA-directed DSA polymerases (RD-DP).thetr ue "reverse trans- criptases"from RNA oncogenic viruses (20, 21) .can be distinguished both on the basis of their intraviral localization as well 8S on their unique ability to make DSA copies from natural RNA templates. RD-DPs from normal cells [Lc e .• avian (22) and mammalian (23-25») can hcveve r be dis- tinguished only on t.he basis of their ability to transcribe DNA copies [rom hete- -:>o l ytDeri cR.~Atemplates.
More detailed information concerning these various classes of re>
plicative DNA pojyeera sea willbe discussed in subsequent sections. In accordance "'ith the newly adoptednomenclature and for the sake of nn tfo rmfty,all DNA polymerases that have been described in the litera - rure , regardless of the terminology used by the authors,vill be referred to as DSA polymerases a, B, and y,on the basis of the distinguishing features outlined in Table II. Also,tenDinal deoxynucleotidyl trans- ferase (TdT) will be briefly discussed for the purpose of comparison.
(B) Reaction Mechanism of Replicative DNA PolY1llerases All replicativeDNA polymerases reportedto date, whether of eukar-yotLe or prokaryotic origin have the aaee basic requirements (16).
xaeety,all eeeber s of this class of enzymes require a template-primer ,the four deoxynucleoside triphosphates,and a divalent cation (either Mg2+ or MD,2+). FurtheI1llOre a 3'OH group on the primer strand is essential,since digestion of theteepIete-p.r feerwithmicroaoceal:nuclease,which pro-
- 8 -
duces 3'-phosphoryltermini, results in the abolition of template activity (16. 26,27,28,29). The imponance of the divalentcations has been shownto reside in their ability to prcecr e the binding of the decxy- nucleoside triphosphates to the enayse (30). and not inpr01llOting the fOIlll8.tion ofth e template-prIme r-DNA pol yme r a s e complex. The latte r function ha s been att ributedto the presenceof Zn2+.which is foundin all pojyeeraees , regard lessof their origin (16.31 -33).
Careful kineticanalysis ofth e ro l e of eacb of the above mentioned cocponent s in the polymerization reaction ,as loIe11as magnetic resonance relaxation studies, have resulted in the elucidation of the reaction mechanism of DNA polymerases. Although much of this work has been carried out ....ithE.coliDSA PolymeraseI.thereisre a s on to believe that the same mechanLsrais operative ineukaryotic DNA polymerases. The proposed mechanism [Scheme III ,modified from Slateret al. (30)]con-
~1stsessentiallyof a twostep process, a pr ree relongationstep (Scheroe III, stepsI-II I ), and a primer trans loaazionstep (Scheme III, steps III-V).
The el.ongationstep consists of the (ollowing events; (1)Aninitial binding o( the polymerase to the primer-templateD!\A through coordination be tv..een the 3'OH group of the primerand·t h e enzyme-bound Zn2+(Scheme III.
step1). (2) Binding of th e deoxynucleosidetr fphosphatea by coordination of the a-phosphory lgroup to the enzyme-bounddivalent cation (MgZ+or MnZ+). This ternary complex is in turn further stabilized by base pairing of the nucleotide to the template strand (Scheroe III, step 11). (3)A nuc Ieo- phil1c attack on the a-phosphoryl group by the 3'OHgroup subsequently occurs with a concerted displacement of pyrophosphate (Scheme III,steps II and III). At this point the nucleotidyltransfer is complete and the
/":-
lllrr H'
m
JJttttt..
1 '''0
Tllff
N
ill-ill-t...
J /';,
lIT!'! \. -
x l .ttlJtt ...
SCHEMEIII
Foranexplana t io n,seete xt[modif ied frOll Slater(It al.
(30 l
- 10 -
~z.ocati<mstep,consisting of the follO\ling. begins: (1)Water ligands substitute for the phosphodiester ligand on the enzyme-boundMn2+ (Scheme III. step IV). (2) Ligand substitution on the Zn2
+at01ll results in the dissociation of the 3'-oxygen atce of the previous nucleotide (now in a phoaphodf eat.er link a g e ) and coordination of the new3'OM group at the end ofth e growing strand with th e enzyme, thus bringing abouttr a n s- location (Scheme III,step V).
Asto whether this latter complex remains stable . and the same enzyme 1llOlecule repeats the same cycle over and over again (1.e.,a processive process). or whether the enzyme is released after a single addition and binds to a different template-primer(i.e •• a distributive process), is at present controversial. For example.Chang (34) and McClure and Jovin (35) claim thatE. aoliDNA pokyce re se I (34, 35) and calfthymus DNA polymerase a and
e
(34)dissociate from the template-primercomplex after a single nucleotide addition, whereas Uyemura(Ital. (36),using E.ooti DKA polymeraseIclaim that the mechanism is p roce s s Ive , l\evertheless.due to the high inefficiency of a distributive mechanis•• it is probably safe to assuae that a processive mechanislll occursin vivo,and that the mechanism is much sore complex than is actually depicted in Scheme Ill.
For example,the polymerizing complex may have otherfactors (I.e.,DNA binding proteins) associated with it that would stabilize the template- primer-polymerase complexand render themechanism processive. The in vitroobservation of a distributive mechanism is certainly insupportof the scae..,hat relaxed requirement for allfour deox)"Tlucleoside triphos- phates ...,hich has often been observed for euuryoticD~Apolymerases (31-39).
- 11 -
III. Characteristics of Various Replicative DNA Polymerases (A) DNA Polymerase a
(a) Basic Distinguishing Features
DNA polymerase a was the first DNA polymerase to be described in a mammalian system (40). The enzyme constitutes the major DNA polymerase in most eukaryotic cells,and may represent as much as 80-90 percent of the totalcellularDNA polymerase activity in activelyproliferating tissues(II ) . Itcan be distinguished from other cellula rDNA polymerases on the basis of its high molecular weight (>10 0 , 0 0 0 daltons), template specificity , andsensitivitytoth i o l blocking reagents. These dis- tingui s hin gfe at u r e s are outlinedin TableII. Table III summarizes some specific propertiesof a-polymerases isolated from various eukaryotic systems. The nomenclature systems used by various workers are also , Ls tcd in TableIII(column 2).
(b) Purification
The low levels of enzyme (approximately1mg/kg tissue ) (I2) evenin highly proliferating tissues. as well as the apparentheterogeneity in size[( 6 2 ) . see also sect ionon Size») ,have greatlyhinderedthe purifi - cation of the enzyme. Nevertheless. substantial purification thro u gh conventional purification procedures has been achieved with preparations from a variety of sources. For example , the cytoplasmic enzyme from rat ascites hepatoma cells has been purified490- and 560-fold (26 , 42a ) ,that from chick embryo to 50-60 percent homogeneity (63) and t.hat.from human KB cells t.o a specific activity of 7,300unit.s*/mg prote In (28. 44a).
In addf r fon , the enzyme from normalhuman blood lymphocyteshas been
* A unit. is1nmoleof labelled deo xynuc Leos.Lde triphosphatein c o r por- ated per hour.
I I
':~ .:.:
, , .
; ;!I h . , . ~I In j" "
II
_Eg
'"
~. .. .
1.
! ~ ~.
.loli
~..
~. . .
j
,
L.I .
• .
I
,
H
'.I
Ii'iz
! I· ,
Ii
z, ~
~i~
:!!~. ! ~ ,
~~i" .
.11.,;g
~,- .. .. · i
i-
i. .
Bl! . · i
".
if , lp iJ jI .j,!.- -- 1 1 1 ..j
s,j,
h
II
r l
5-.jii
~Ii P
Hr
i- i-·-H BHU
:-P
itII :- Hii
lEU,-
Ii II
jl H jl j ~ " 13
- 13-
purified 220-fold(51),and that from BHK-21!C13 cells (59) 154-fold throughasimpleextractionprocedu r e fo llowe dby ion exchange chroma to- graphy on phosp hoc e llulos eandgel filtra tion onSephade xG 100.
(c) Properties (1) Telllf?lateSpecificity
Earlyvcrk with DNA pcdyee ra se a re v e a l e dth a t relative to nativeDr-A, denaturedDNA was th e preferred templ ateforD~Asynthesis (64). The ability of denatu re dDNAtoac t as aneffi c i ent templatewas la t er shown to be dueto the presence of "hairpin"structures providing the 3'OHprimer groups fmpe ratIve forth e initiation of DNAsynt.hesis (65, 66). In genera l the most ef fi c i enthete ropo IyaerIc DSAtemplates are nativeDNApartially di g e s t edwi th p ancreatfc DNaseI (a l s o referre d to as"ac ti v a t ed" DNA) (26, 28,37, 67-70),and"g a p ped "D~ A(exonuclease IIItre a t e d"a c t i v a t e d"
DNA) (28.55). Surp ris inglyhowever,Sed\.licket al. (28) have observed that chen"a c t i v a t e d " D~Ais converted to"g a p pe d" DNA (vf r h very little increasein the numberof 3'OH group s ).therewa s no increaseinits ef- ficiencyas a te mplat e . Thisis probablyrelatedtotheinabilit yof DNA polymerase a totr a v e r s e long gaps,consistentwith anin vitrodis- tributive mechanism [see sect.ionon Mechanism, 11(B)].
Native DNA has also been reportedto be used by DNA polymeraseQ. ho ....ever .thisability has beenfoundto decrease \.lith anincrease in the degree of pur ificationcVth e eneyeeunder investigation (37. 71 , 72). Presumably the use of native DNAis due at least in part to the presence in t.he early purificationsteps of contaminating endonuclease activity capable of "activating" nat iveDNA and thusre n d e r i n g it an efficient template .
The syntheticdeoxyhomopolymers.pol y (dA) and poly (dT), investigated
- 14 -
as templates for calf thymus DNA polymerase a,are used only in the pre- seace of complementary oligonucleotide primers (73),while in the case of poly (ecj,no primer is required since at neutral pH this polymer is capable of forming"h a i rp i n " structures to provideth e initiation sites (73). The synthetic cop oIye er polyd(A-T)isus e djust as efficiently as "activated "DNAby polymerase a from bone marrow (5 2 ). Th eem:yme has also been reported to use pckydeoxynucLe o t fde templates initiated with ol igo ribonucleo tides(6 2 . 74. 75) sawe ll as RNA-p r1uledDNA as template (76). DNA polymerase a vill not however use polyribonucleo- tides aste mpl a t e s when initiated wit holigonucleotides (28.37,62. 70.
14, 7 5 ,77) .
Certain parameters to be kept in mind when testingpoly- and oligonucleotides as te mp l a t e- pr i mers are: the ionic strengt hof the medium , th edi v a l entcati on concent rationandthe temperatureunder which the assays arecarriedout since allof these factors affectth e stability ofth e oligomer-polymercoepIexea (78). In addition the tem- plate preference of DNApolymerase a changes with the assay conditions (72), and also with the degree of purification of the enzyme - presumably because of the eliminat.lof\to various degrees,of contaminating nucleases at various stages of purification(37).
(11) Divalen tCationRequirement
AllDNA po Iyce re se s reportedto date,includ ingDNA polymerase0, display an absolute re qufreeencfor a divalent cation for activity. This requirement is usually met by magnesium,vhen "activated"DNA is used as the template. Although manganese can to some extent replace aagnesium (43,45).it is much less effective. Optimalconcentrations of Mg2+
lie in the range of1 t.o 12 m.\i depending onassay condit.ions and the
- 15 -
source of the polymerase(26-28 , 45, see also TableIII). On the other hand, optimalMn2+concent rat io ns aremuchlower, inth e range of 0.05- 0.40mM,also dependingon the assay conditions andthe sourceofth e polymerase(4 3,45).
Divale n t cationoptima are difficult,if not impossible,to assess since a number of parametersofth e DNA polymerase assay influencethese values. Among suchfactors arethena t ure andconcentration of the tem- plate, the concentr at ionsof th edeoxynucl eoside tr Iphosphe t.e s (53, 79, 80), andthe pH (52). Thus the widera n g e of divalent cation optimare- portedmay be due to the variationsin assay conditions used in different la bo r a t o r i e s . Craig andKe ir(80 )ha v e pointedou t that the higherthe concentration of template and/or deoxynucleosidetriphosphates,the higher is the optimum concentration of the divalent catio n. This is at least partiallydue toth e chela tingactionof the triphosphates , as wellas the binding ofth e divalentcationsto the nucleic acidtem- plate, especially at higherpHva l ue s. Contraryto this expectedpH effect, DNA polymerase c frombone marrow exhibited an increase indi- valentcationcptLmum uponde c r e a s i n g the pH ofthe assay (52). Enzymes incapableofusingMn++ha v e also been reported. For example,DNA poly- merase(lfrom the nuclearmembrane-chromatinfraction of rat ascites hepatoma cells was foundno t to catalyzeDNA synthesis in the presence of Mn++(2 7,42a,54) . This is probably not a unique feature of the enzyme but may merely re f l e ctth e assay conditions used.
(ii i ) Effect of Monovalent Cations
Monovalentcations have been reported to both inhibit (2 7 , 41,42a, 47,48 ,51,54 , 59,63,81 , 82),as wel las stimulateDNA polymerase activity (50,52,58,83 ,84 ) . Thena t ure ofthe ttechanism for this
- 16 -
stilllulation is coepIe x , and probably involves both the eneyee and the template. Somewor k e r s have found thatthe stimulationis specific for only certaincations(50. 58, 83). Moreover,the effect has been shown to be influenced by the type of teep Late used(518,63). Chick embryo DNA polyJDerase(Iwas in h i b i t e d inth e presenceof sal twhen "a c t i v a t e d"
DNA was used asthete DlPl a t e . whereas with poly(dA)·poly(dT) the optiaUlD concentra tionof KCl was 40 mM (5la ,63). Wi th re ga r dto specific ion effects,Laza r u sand Kitro n(58, 85) haveshownthat th e cytoplasmic DNApc'l ymer a aeC1ac ti v ity frombaby ha ms t erkid n e y (BRK)cells ....as en- hancedbymonova l e n tcat i ons in the or d e r :t<o"H
4
+
>K+>Na+>Cs+>t.i", ....iththe latte r cationbeingin h i b it ory. The effect seemedtobe rel at e d to the crystalline radii ofth e cations, andit ....as concludedth a t a crystalline radius of 1.4 2 was optimal for stimulation (58). Theya190 eade the interesting observation that the sulfatesalt of attIIOOniUJllions IoI&S substantially less sti... l&tory than the chloride salt . However . this My be dueto the inhibitory side effects of sulfate ions on enzymes 1n- volved inphosphoryl transfer (58).(1v)~
Ingenera lthepH optimulll of DNA pod yeer-a s e0.lies Dear neutrali ty.
Forexample,poj ymera a e sa from ratthymus (83) . rat intestina l mucosa (47,48) ,rat asci t e s hepatoma cells(26,42a ,54),chick embry o (51 a, 63), card iacmuscle (49),no r malhumanlymph o c y t e s (51),andBHK-21/C13 cells (59),displayedpH optima. be tween6.8and 7.65. I t is important to note however , th a t the optililUli pH depends on a varietyof parameters, such as the nature of the template (51,53) and buffer (27,42a,54,55. 58),as well as on the nature and concentration of the divalent and IIOno- valentcation used (52). In partic u la r ,DNA pol)'1llE'rase a frailhepatoma
- 17 -
LF cells exhibited a pH optimum in the range of 7 to 7.4 when assayed with the template poly (d(A-T)·d(T-A»). pH 7.6 to 8.4 "'ith poly (dC), and 6.8-7.8 with heat-denaturedD~Aas template (53). With regard to the nature of the buffer, the enzyme frOlll rat ascites hepatoma cells had a pH optilllUIII of 7.0in potassiUlll phosphate.7.8 in Tria-HCI,and 9.5 in glycine-NaDH buffer (55). Anincrease in pH optimum has been observed in the case of D~Apolymerase(Ifrom bone marrow (52) when the divalent cation concentrat ionin the reaction mixture was decreased. This ",as also found to be the case when the monovalent cation concentration was altered (52). Thusit appears thatthe determinationof an absolute pH optimum for the enzyme is a difficult task due to the influence exerted upon this parameter by other components normally present in the assay systees , This would explain the wide range of pH optilll8 reported from the various laboratories engaged In the characterization of DSA po1ymerases.
(v) Isoelectric Points
DNA polymerase(IfrOlll a variety of sources has been shown to be sn acidic protein. This conclusion is based on the relative affinity of the enzyme for ion exchange resins such as DEAE-ce1lulose and phospho- cellulose (44a). In particular,the r scej ect r rc point (pI) of DNA poly- merases a frOlD normal human lymphocytes (51) ha s beendirectly shown, by Isoelectric-focusing, to have a value of 4.5. This valueis probably too low, however,since any nucleic acid that may be associated with the poly- merase must be eliminated prior to detennining pI's by tecefect r r.c- focusing; otherwise the pI'sobtained will be those of a protein-nucleic acid COmplex,and hence too acidic (86).
- 18 -
(vi) Size
DNA polymerase0.from various sources (62. 70, 84. 87-93) appears to be heterogeneous in size [see Tables III (column 4) and IV]. This hetero- geneity may be due to aggregation (44a, 52, 78. 94), proteolysis (12. 92, 95), association with other proteins of the replicative complex (37, 62.
70, 78,87,88, 91, 96). or merely association with nucleic acids (91). In general. the size of the enzyme is in excess of 100,000 daltons al- though a polymerase a of 87.000 daltons has been reported inDrosophila melanoqaet-er(79). The variety of sizes observed for the "large" DNA polymerase from a numbe r of sources, and the molecular species detected under various dissociation conditions are shown in Table IV. The size of some of the species detected under dissociating conditions indicates the possibility of confusion between DNA polymerases a and
e
when identification is made on the basis of molecular ve Lght s alone. Ap- pa r-entLy , some of the large polymerases reported are merely an aggregate form of DNA polymerase 8 or a mixture of both DNA polymerases a and 8 (see comments in Table IV).A lack of correlation between the molecular weights as estimated by gel filtration and by sedimentation velocity analysis has been observed (62, 70, 109, 110). Hoveve r,this may be explained by the apparent asymmetric shape of the DNA polymerase molecule as suggested by Holmes and Johnston (l09). These authors determined that an axial ratio of 10 to 1 for DNA polymerase a of rat liver would explain the discrepancy in apparent molecular weights. The same explanation was offered by Caruso et al.(110) for Dl\A polymerase a of Xenopus laeviewht.ch behaved as a 300.000 dalton species by gel filtration, and as a 145,000 dalton species by sed teenc at tce velocity analysis in glycerol gradients.
- 19 -
- 20 -
The ionic strength at which the sedimentation coefficients and the 1IlO1ecularweights are estimated also influences the est1lllated size. For example, at an ionic strength below 0.07, DNA polymerase a aggregates to 10.75 (52, 94, 111);at physiological or higher ionic strengths,hoveve r, the enzyme has a sedimentation value of 6-85 (14,52,78). It has also been claimed that the size of the enzyme from calf thymus can range from 130,000 to 450,000daltons depending on the conditions to which it is exposed (78).
The heterogeneityof calf thymus DNA polymerase0.has been studied in detail by Holmeset al. (62,92,93). These workers have resolved a partially purified enzyme preparation into three species (denoted as enzymes A, B,and C) on a DEAE-cellulose column (62). Enzyme A has been shown to have a sedimentation coefficient of 85,enzyme B 5.25 ,and enz yrae C 7.25 ,corresponding to molecular weights of 200-230,000.
100-110.000,and 155-170,000 respectively (12,62). The relative amounts of these species varied from one preparation to another,and furthermore, In bl,Jllle preparations two A species (denoted as Al and A
2) were obtained (12. 93). EnzymeA could be converted to the C form by 2.8M urea treat- ment (93) and enzyme B has been suggested to probablyrepresenta pro- teolytic degradation product of enzyme C (12. 62, 92,95). If poly (dA)'(dT)lO was usedas the template instead of "activated" Ol.;A. an additional species (enzyme D, 6.6-75 or 140-150,000 daltons)was detected on the DEAE-cellulose column (62).
With regard to the subunit structure of DNA polymerase0:.Holmes et al. (62) have conc Iuded that the 85 (enzyme A) species is made up of a 55,000 dalton subunit associated with an active subunit of 155,000 daltons (enzyme C). This finding is consistent with the observation by
- 21 -
Sedwicketal.(44a) (or polymerase0 (rolll human KB cells, except thatth e s e authors reporteda subunit size of 77,000daltons. More re c e n t l y, Matsukageet al. (112 )havesheen tha tpolymerasea of mouse myelom a consistsof twosubunits,one of 150,000 an d theot he rof 60,00 0 dalton s.
Discrepanc iesinsubunit size betweenenzymesfr omhumanKBcells , and calfth ymus oreocse myeloma are probablydue toth e use of ecn- hOlllOgeneous enzyme preparations;before arriv ingatany final conclusions it is essentia l that hoeogenecua enzyme prepara tionsbe obtained.
(d) Subce ll ula rLocalization
Bot hea rl y (113),aswell as rec e n t stu di e s (37,43,69,95) directed at determiningthe subcellu l a r lo c a l i za ti on of DNApolymerase a are in conflict with the presumedsite of action ofthe enzyme - that is,the _jor portion ofthe DNA pol)'Uleraseactivi tyin mammalian cells is usually re c o v e r e d in the cytoplasm. Explanationshave been offered forth is , namely,ithas been suggested th atthe finding ofth is activ i ty i.n the cytoplasmmay be due to leakage fromnuclei duringtheir isolat ion, oc that it may representdenovocytoplaslllicsynthesis of enzyme to be translocatedto the nucleusvb e u needed forDNA synthesis(114). Although a number ofinvestigators have failedto detectany DNA polymerase a activityinth e nucleus (37 ,43, 69, 95,127, 128 ) , other workerswere successful in doing so (28 , 41,68,125,126). Chang andBollum(15,37) ha v e shownho...rever that removal of the nuclea r outer membraneyields nucleidevoi dof DNA polymerasea, and thatfail ureto relllOve the outer membrane yields nuclei displayingpolyuerase0 activity.
Non-aqueous isolation methods fornuclei have revealed that DNA poly- ee reae0 could indeedbe found in nucle i (7,ll5- U n . Ithas been shown, eithe r throughtheuse ofnon-a q ue ou s isolat ionmedia (120),orthe use
- 22-
of 30%glycero l supp lementedwith4 tnM CaC1
2with no buffer (118-119), or throu gh enucleation of cells by treatmentwith cytochalasinB (121), that as much as 90%of the total DNA polymerase a activity can be found in the nucleus (118-121). Thusi t appears that the finding of DNA poly- merase(lin the cytoplasmis mostli k el y due to itsle a ka ge fromth e nucleus, and thus the paradoxwi t h regard to subcellular localization and function has beenre s olv e d.
The possibilityth a t DNA synthesis may be initiatedat the nuclear me mbranearoused interestin tryingto locate th epo l yme r a s e u at such asi t e . Thus,the enzyme has been reportedto be associated with the nuc l earmembrane-chromatinfraction (54, 122,123), but it is not clea r whetherthe enzyme is boundonlyto the DNA or nucleoproteincomplexes (123), or whether it is an integralpart of the membrane itself. The enz yme has alsobeen reported to be associated withsmooth microsomal ceebren e s (4 2 . 43,54, 124 ) .
(e) Inhibition Studies
The possibilityof using inhibitors as tools for distinguishing various DNA polymerasesand for determiningthe biological function of DNA po Lymerase s , as well as their potential use for cancer chemotherapy has stimulatedmuchof the work directed at exploringthe effectof vari ous in h i b i t o r s on DNA polymerases. A list of some of the inhibitors investigatedfor their effect on DNA polymerase c is given in Table V.
With regardto theirpossible use in distinguishing DNApolymerase a frolllother DNA pcfyee resee, th e sulfhydryl blocking reagents,N- ethylmaleimide(NEM) (26,27. 42. 42a. 48,51.51a. 52. 55. 57-60, 84, 88,97-99), p-chloromercuribenzoate (p-CMB ) (26.27, 41, 42, 43,44. 48.
5la. 55. 84,99,122),and p-hydroxymercu r ibenzoate(p-OHHgBz) (5 3 ),
- 23- TABLEV. In hib i to rsofDNAPo l yme r a s e e
INHIBITOR SOURCEOFPOLYMERASE fo,'EM. BHKcel ls (+) ....
Rat ascites hepatoma(+) Rat intes tina l mucosa (+) Bone marrow (+ ) Chick embryo (+ ) HeLa cel lS3nu clei(+) Developing chick he art (+ ) Chineseha ms t er cel ls(+) Culturedmouse L929cells (+) Normal human bloodlymphocytes
(+) Mouse myeloma (+) Morrishypatoma (+)
pCMB Rat ascites hepatoma (+)
Adult rat liver (+) Rat intestinal mucosa (+) Chick embryo (+) HumanKBcells (+) Culturedeouse L929 cells (+) Hor rishepatoma (+)
Calf th)"1IlUS nuclearmembrane (+) pOHHgBz LF hepatoma(+)
Heparin BHK cells (+)
Ethidium Bromide ger Icufocyr es (+) Bone marr",", (+) HumanKB cells (+) ActinOQlycinD Bone marrow(+) Acridine Orange HumanKBcells (+) Nalidixic Acid Rat intestinal mucosa (+ )
Bonemarrow (- ) araeTP Developingchic kheart (+)
Rat liver (+)
Haem Reticu 10cytes (+)
Bone marrow(+ ) AF/013 Re rfcuIo c ytee (+)
Bon~marrow (+) NaPPi Ratasciteshe p a t oma (+ )
HumanKBcells (+) Methanol, Ethanol House JDyeloma (st i mu l a t ed)
and Isopropanol Rat ascites hepatoma (+)
REFERENCE 58, 59,97 ,98 26, 27, 55,99, 42a 48
52 51 60 27 57 84 51 88 42
26, 27,55,42a, 99 43,41
48 51 28, 44a 84 42 122 53 58,59, 97,98 50 52 28,44a 52 28,44a 48 52 27 129 50 52 50 52
26, 27,55,42a,99 28,44a 88
26, 27, 55, 42a,99
For list of abbreviationsreferto page (xi)-(xii).
"+"indicates inhibition and"-" indicates no effect.
- 24-
enable us to distinguish DNA polymerase CI fromB. As noted in Table V, polymerase a is ex t remelysensitive to these compounds whereas DNA poly- meraseB,as we shall see later,is relatively resistant.
Heparin has alsobeen re p o r t e d to inhibit DNA polymerase a from BHK cells (58, 59, 97,98). Furthermore,haem andthe ri f a mycin derivative 3-formyl rifampicin SV:O-n-oc tyloxime(AF/013) have been reported to in- hibit the polymerase CI from reticulocytes (50) andbone marrow (52).
Othercompounds reportedto havean inhibitory effecton DNA polymerase a are: sodium pyrophosphate (26-28 ,42a,44, 55,99) l-B-D-arabinofu r- anosyl cytosine 5'- triphosphate(araC!P) (27), and isopropanol,methanol, and ethanol (26, 27,42a,55,99). However,in the case of the latter compounds,theireffect on the polyme raseCI from mOuse myeloma was of a stimulatoryratherthan of an inhibitory na t u r e (88).
Less specificinh i b i t o r s , exertingtheireffect by binding toth e template,consist of compounds such as ethidium bromide(28,448,50, 52), actinomycinD(52),nalidixic acid (48, 52) ,and acridineorange (28,44a).
(f) Biological Function
The most un a mb i gu o us approachindetermininga function for a particula r enzyme (e.g., DNA polymerasea), is to isolatea mutantlacking or havfng a defective enzyme,andcorrelating this deficiencywiththe loss of a specific biologica l function (e.g. , DNA replication). Suchan approach isre l at i vel y simplewhen dealingwit hprokaryotes,but with eukaryotes it is extremelydifficultto obtain such mutants. Due to this difficulty ,alternate approaches have beentaken in the attempt to assign a biologica lfunction to DNA polymerases. Such approaches have consisted ofcomparisons between the relative le v e l s of differentDNA polymerase activi tiesandthein vivorates of DNA synthesis intissues
- 25 -
and cell cultures challenged to proliferate,comparisons of the relative levels of different DNA polymerases in tissues from quiescent. to actively proliferat.ing stat e s,and t.he disappearance of the enzyme in t.erminally different.iat.ing syst.ems, as well as a comparison in properties of t.he various DNA polymerases and t.hat. of an act.ively proliferating complex (i.e.,effect of unwinding proteins on polymerases as well as their abilit.y to use RNA-primed DNA templates). All such evidence is circum- stantial and does not le a d to the assignment of a Fu n ctLon with cer-ta fnt y,
A series of studies making use of such indirect approaches have im- plicated DNA polymerase a with DNA replication. A positive correlation between the level of activityof this enzyme and thein vivora t e of DNA synthesis has been reported in the follOWing systems: regenerating rat liver (42, 95,127,130). mouse L cells (95),HeLa cells (60,131, 132), BHK cells (133). chemically induced tumors (78,95),PHA-stimulatednonnal human lymphocytes (51,134), tissue culture cells at various growt.h rates (8),erythropoietine-inducedmouse spleens (135),and phenylhydrazine- induced reticulocytosis in mouse spleens (78, 95). In addition. it has been shown that terminally differentiating chick myoblasts lose their DNA polymerase0 activity while they retain polymerase 6 (53a). Although incapable of undergoing further mitosis. the myotubes retain the ability to repair DNA (53a). Furthermore. it has been observed that a decrease in DNA polymerase a activity occurs during development of mouse embryo brain (136). All the results obtained with the above systems clearly indicate that the level of DNA polymerase a varies according to the rate of cell proliferation and thus parallels DNA replication.
A temperature-sensitive mutant of the smut fungusUstilago maydis having a heat labile DNA polymerase activity has beenfsoj ar ed (137).
- 26 -
This organism apparently ha s no DNA polymerase 6 (138, 15 3 ) so all activity is duetopol yme ra se II. Consisten twdt.h a replica ti ve ro l efor DNA polymerase a. the temperat u re -sensitivemutant lo s e sthe abil it y to replicateits nuc lea r DNA at th e rest r ict ive temperaturewh ileret a i n i n g the abilityto rep l icateitsmitochond ria l DNA[PoUrrr au,personalcom- munication to Bankset: al. (138)
J.
Generally speaking , replicationisacomplexprocessinvolving the participationofnot onlyDNA polymerase,but al s o of severalother accessory proteins (139). As inthe case of prokaryotic systems,a number of reportsdealingwith prot e i n fac to rshaving astimu la toryef- feet on DNA synthesis in eukaryoticsystemsha v e appeare d in th e literature (140- 14 6 ). I tis interestingto note that wh e n such stimu latory factors werein v e s t i ga t e d wit hrega rd to their effects on in vitroDNA synthesis,in the presence ofeither polymerases II and6,a substant ial stimulation of activitywas observed with the polymerase II only andno such enhancement was evident with 6 (140, 141, 143,146) or mitochondria l DNA polymerase (14 0 ) .
The observation in both bacterial(147 -14 9 ) and mammalia n (74.150 - 152) systems thatRNA servesas a primer forin vivoDNA synthesishas led to a comparative study onthe ability of bo t h polymerasesII andBto make use ofR.~A-primedDNA templatesfor DNA synthesis (62. 74-76). The overall conclusion·fromeuc h invest iga tions is thatonly DNA polymerase
II possesses the abili tyto useR.~A-primedDNA templates forDNA synthesis.
consistentwi t h a possible replicative function for this enzyme.
Thus there exists a great dealof evidencethatDNA polymerasea is the replicating enzyme,even though much of this evidence is circumstantial.
