Assemby of DNA Replication Proteins_Todd L Capson

8/8/2019 Assemby of DNA Replication Proteins_Todd L Capson

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Cel l , Vol . 65, 249-259, Apri l 19. 1991, Copyright 8 1991 by Cel l Press

Protein-DNA Cross-Linking Demonstrates StepwiseATP-Dependent Assembly of T4 DNA Polymerase andIts Accessory Proteins on the Primer-TemplateTodd L. Capson, t Stephen J. Benkovic,and Nancy G. Nossa lS*Depar tment o f ChemistryPennsylvania State UniversityUniversity Park, Pennsylvania 16602*Section on Nucleic Acid BiochemistryLaboratory of Biochemical PharmacologyNational Insti tute of Diabetes and

Digestive and Kidney DiseasesNational Insti tutes of HealthBethesda, Maryland 20892

SummaryT4 DN A polymerase, the 44/62 and 45 polymerase ac-cessory proteins, and 32 singlestranded DNA-blndlngprotein catalyze ATP-dependent DNA synthe sis. UsingDNA primers wlth cross-l lnkable residues at specificpositrons, we obtained structural data that reveal howthese proteins assemble on the primer-template. Withthe nonhydrolyxable ATP analog ATP+, assem bly ofthe 44/62 and 45 proteins on the primer requires 32protein but not polymerase. ATP hydrolysis changesthe position and intensity of cross-l inking to each ofthe access ory proteins and al lows cross-l inking ofpolym erase. Our data indicate that the initial bindingof the three access ory proteins and ATP to a 32 pro-tein-covered primer-template Is fol lowed by ATP hy-drolysis, binding of polymerase, and movem ent of theaccess ory proteins to yield a complex capable of pro-cessive DNA synthesis.IntroductionDNA synthesis is accompl ished by mul t ienzyme system swhose componen ts interact to ensure rapid and accurateDNA repl ication. Structural information on these sys tem sis crucial to understanding their function, but such informa-tion has been diff icult to obtain, owing to the weak associa-tions between some o f the proteins and the lack ofsequence-specific binding by enzym es that move with therepl ication fork. The DN A repl ication syst em of bacterio-phage T4 includes T4 DNA polymerase, the genes 44/62and 45 access ory proteins, and the gene 32 single-stranded DNA -binding protein, wh ich are all required forsynthesis on the leading and lagging strands (for reviewssee Nossa l and Albens, 1983; Richardson et al., 1990).The access ory proteins increase the processivi ty of thepolymerase in an ATP-dependent reaction. T he relativelysimple T 4 syst em has served as a model for more com plexrepl ication sys tem s that include the same types of compo-

TPresent address: Nat ional inst i tutes of Heal th, Bui lding 8, Room2A-15, Bethesda, Maryland 20 992.

nents (Tsurimoto and Sti l lman, 1990). Howe ver, very l itt leis known about the structure of the T4 proteins on aprimer-template, the manner in which they assemb le, orthe role of ATP hydrolysis in this process.

The T4 polymerase access ory proteins are thought tofunction as a clamp that secures the polymerase to theprimer-template. Depending on the avai labil i ty of dNT P,the access ory proteins stimulate either the polymerase orthe 3-5 exonuclease activi ty of the polymerase. The 44and 62 proteins are isolated as a tight complex that hasa weak DNA-dependent ATPase activi ty that is stronglystimulated by the 45 protein (Piperno et al., 1978; Maceand Albert% 1964; Rush et al., 1989; Jarvis et al ., 1969b).44 Protein alone retains the ATPase of the 44162 complexbut is poorly stimulated by 45 protein (Rush et al., 1989).Aside from the tight complex of the 44 and 62 proteins,the physical associations between the polymerase, theaccess ory proteins, and 32 protein are weak . Protein affin-i ty chromatography has helped to define pairwise physicalinteractions between the proteins (Form osa et al., 1963;Alberts et al ., 1983). The 44/62 protein can be isolated ona 32 protein-covered primer-tem plate after gel filtration,but 45 protein remains bound only i f ATP and polym eraseare also present (Richardson et al., 1990). It has not beenpossible to simultaneously footprint the access ory pro-teins and polymerase (Munn, 1966). In addition to increas-ing the polymerase processivi ty, the access ory proteinsstimulate duplex unwinding by the T4 gene 41 hel icase(Venkatesan et al., 1982; Richardson and Noss al, 1989a),are required for RNA primer synthes is by the T4 primase-hel icase i f the T4 template is covered with 32 protein (Rich-ardson and Nossa l, 1989b), and are involved in the tran-scription of the late T4 genes (Herendeen et al., 1969,1990).

Photochemical cross-l inking can detect weak protein-DNA interactions and is particular ly useful in locating co m-ponents of multisubunit enzym es or multienzym e com-plexes. Several different cross-l inking methods have beenused to study interactions of RNA and DNA polymeraseswith their respective substrates (Dissinger and Hanna,1990; Bartholomew et al., 1990a, 1990b; Evans and Haley,1967; Catalan0 et al., 1990; Hockens mith et al ., 1966).To study the assembly o f T4 po lymerase, the accessoryproteins, and 32 protein at the primer terminus, we havedesigned a primer-template sys tem in which the positionof a cross-l inkable aryl azide can be moved stepwisethrough a30 base region of the primer by selective elonga-tion of the primers with different combinations of dNT Ps.We have carr ied out cross-l inking experiments with al l f iveproteins in the presence of ATP or ATPyS . Our data sug-gest that the ini t ial formation of a complex of the accessoryproteins and 32 protein with the primer terminus requiresbinding, but nothydrolysisof, ATP. This is fol lowed by ATPhydrolysis, movem ent of each of the access ory proteins,and the addition of polymerase to yield a T4 holoenzymecapable of processive DNA synthes is.

Todd L. Capson, materials for application for Senior Program Officer, TRAFFIC North America


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Figure 1. Primer-Templates wi th Photoact ivatable Thymidine Ana-logs Used for Cross-Linking Experiments(A) l l /PO-Mer used for cross-linking studies wi th the T4 polymerase.The sequence of bases in the 29mer al lows elongat ion of the 1 I -merto the 12-mer wi th dCTP, to the 1Cmer wi th dCTP and dATP, to the17-mer wi th dCTP, dATP, and dGTP, and to the 29mer wi th al l fourdNTPs.(B) 34-Mer-@X174 duplexes used for cross-linking experiments wi ththe T4 polymerase, accessory proteins, and 32 protein. The two 34-mers used were of ident ical sequence but di f fered in the placement ofthe cross-linkable residue. The 34(4)-mer and the 34(20)-mer havethe cross-linkable residue 4 and 20 bases, respectively, f rom the 3primer terminus.

Resul tsCross-Linking of T4 DNA Polymeraseto the 11/2O-Mer SubstratesPrevious studies from one of our laboratories employedDN A substrates that contained reporter groups near the 3terminus of the primer to study DN A polymerases (Gibsonand Benkovic, 1987; Catalan0 et al., 1990). We used the1 /20-mer shown in Figure 1A to cross-l ink T4 DNA poly-merase in the absence of additional T4 repl ication pro-teins. The primer contains a modified thymidine residue towhich a Sazido-2-nitrobenzoyl moiety is attached via a3-carbon tether, placing the cross-l inkable moiety withinor close to the major groove of the DN A hel ix. Irradiationof the cross-l inkable residue with ultraviolet l ight at 302 nM(photolysis) results in formation of a nitrene that can formcovalent adducts w ith adjacent proteins (Bayley, 1983;Bayley and Knowles, 1977).

In the 11-mer, the modified thymidine is two bases (-2)from the 3 terminus. The autoradiogram in Figure 2Ashow s th at the 11 -mer can be elongated to the 12-, 14-,17-, and 20-mer in the presence of the appropriate combi-nation of dNT Ps, which positions the cross-l inkable resi-due at -3, -5, -8, and -11, respectively. After elongationof the primer, the reaction mixtures were photolyzed,boi led, and the samples were analyzed on a denaturing

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Figure 2. Elongat ion of the 1 /20-Mer by T4 DNA Polymerase andCross-Linking of the Polymerase by DNA(A) Autoradiogram of a DNA sequencing gel showing elongat ion of the5~[Pj l l -mer primer (lane 1) to the 12-, 14-, 17-, and 20-mer by theaddi t ion of dCTP (lane 2) dCTP and dATP (lane 3) dCTP, dATP, anddGTP (lane 4) or dCTP, dATP, dGTP, and dTTP (lane 5) respect ively.(B) Autoradiogram of a 10% SDS protein gel showing cross-l inkedpolymerase. Lane 1 is DNA alone and lane 2 is f rom a DNA samp lethat was photolyzed both before and af ter addi t ion of the polymerase.Assays for lanes l -5 contained 0.1 mM EDTA and no MgC& or dNTP.Lane 3, 1 /20-mer (cross-l inkable residue at -2) and 200 nM polymer-ase. Lanes 4 and 5 are ident ical to lane 3 except that polymerase was400 nM (lane 4) or 600 nM (lane 5). Assays for lanes 6-9 contained 7mM MgCI, and 200 nM polymerase and dCTP giving the 12/20-mer(lane 6); dCTP and dATP (14/20-mer, lane 7); dCTP, d ATP. and dGTP(17/20-mer, lane 6); or dCTP, dATP, dGTP, and dTTP (20/20-mer, lane9); with the cross-linkable residue at -3, -5, -6, a nd -11, respectively.

SDS protein gel to detect covalent polymerase-DN A ad-ducts. T he autoradiogram of a protein gel in Figure 26shows that the 1 1-mer ( lanes 3-5), 1P-mer (lane 8) and14-mer ( lane 7) are capable of forming covalent cross-l inkswith the polymerase, but the 17-mer and 20-mer ( lanes 8and 9) are not. We conclude that polymerase is in closecontact with the fi rst 5 to 7 bases of the primer.Cross-Linking Experiments with T4 Polymerase,Access ory Proteins, and 32 ProteinTo determine the positions of polymerase, the access oryproteins, and 32 protein together on the primer, we usedcircular ss@ X174 DN A as a template and the 34-mer co m-plementary to nucleotides 808-841 as the primer (see Fig-ure 1 B). We made two versions of this 34-mer: 34(4*)-mer,in which the modified thymidine (T*) is 4 bases removedfrom the 3 end (-4), and 34(20*)-mer, in which i t is 20bases removed (-20). The T residue in the 34(4*)-mer isfol lowed by three Ts so that, in the presence of dTTP , i t isprotected from the exonuclease activi ty of the polymerase.The template sequence al lowed the polymerase to elon-gate the 34-mers by 5 residues with dlTP and dCTP, andby 10 residues with T , C, and G ; this mov es the cross-linkable residue from -4 t o -9 and -14 with the 34(4*)-mer, and from -20 to -25 and -30 with the 34(20)-mer.We chose asequence where dATP w as the last nucleotideto be added, since the ATPase activi ty of the 44 proteinalso uses dATP as a substrate (Mace and Albert% 1984);this allowed us to asses s the effect of ATP and AT P ana-


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1 2 3 4 5 6 7 1 2 3 4 5 6 7

Figure 3. Elongat ion,p f the 34(4) P rimer by T4 DNA Polymerase, the44/62 and 45 Accessory Proteins, and 32 ssDNA-Binding Protein(A) Lanes 1-3, ATP(PL ul t rapure); lanes4-6, HPLC-puri f ied ATP; lane7, no ATP.(B) Lane 1, no enzymes; lanes 2-4, ATP (PL ult rapure); lanes 5-7,ATPI .

In the presence of dlTP ([A] , lanes 1 and 4; [B] lanes 2 and 5) thepolymerase can idle over three dTM Ps with no net elongat ion of the5-[=P]34-mer (see Figure 1 B); degradat ion products f rom the 3+5exonuclease act ivity of the T4 polymerase are evident below the34-mer. The 34-mer can be elongated to the 39-mer in the presenceof dITP and dCTP ([A] , lanes 2 and 5; [B] , lanes 3 and 6) and to the44-mer in the presence of dITP, dCTP, and dGTP ([A] . lanes 3,6, and7; [B] , lanes 4 and 7).

logs on the cross-l inking reactions. The large circular tem-plate was used to prevent degradation by the exonucleaseand to serve as a trap for proteins bound nonproductivelyon the single-stranded portion of the template.

To minimize problems resulting from contamination ofone nucleotide with another, w e determined the lowestconcentrations (25 FM ATP, 20 uM each dNTP) necessaryfor access ory protein-dependent DNA synthes is. Controlexperiments with Ml 3mp2 annealed to a 84 base primer,which included a 50 base unpaired tai l (Richardson et al.,1990) showed that these low nucleotide concentrationsdid not appreciably decrease the rate of copying of the 7.2kb single-stranded template, al though they did preventstrand displacement synthesis from the resulting forkedduplex.

In al l of the cross-l inking experiments with the T4 replica-tion complex, the reactions were carr ied out with an ex-cess of the polymerase and access ory proteins, with 32protein sufficient to cover about 50% of the template,which is the optimal ratio of 32 protein to DN A. The en-zym es and DN A were incubated for 8 s, an al iquot of thereaction mixture was removed for analysis of the DN Aproducts, and the remainder irradiated for 1 min. Figure 3show s that in the absence of UV l ight, the 34(4)-merprimer w as elongated to the expected 39- and 44-merproducts after 8 sat room temperature. The same produc tswere observed after 1 min (data not shown). The 47-merapparent in lane 3 of Figure 3A is due to the presence ofsmall quanti t ies of dATP in the ATP. Puri f ication of theATP by high pressure l iquid chromatography (HPLC) re-sulted in decreased amounts of 47-mer (Figure 3A, lane8). There is residual 34mer in al l of the lanes that hasnot interacted with polymerase; although polymerase ispresent at a higher concentration than the primer-tem-plate, so me o f i t is bound to the single-stranded template.

The effect of the polymerase access ory proteins is ap-parent even under these conditions of l imited DN A synthe-

sis. In the presence of ATP and the access ory proteinsthere is no accumulation of products between 35 and 38bases long, in addition to the expected 39-or 44-mer (Fig-ure 3A, lanes 2, 3, 5, and 6; Figure 38, lanes 3 and 4).These incomplete products do accumu late i f ATP is omit-ted (Figure 3A, lane 7) is replaced by ATPyS (Figure 38,lanes 6 and 7) or if either the 44162 com plex or 45 proteinis omitted (data not shown).T4 DNA-Re plication Protein Complex Formedwi th ATPPolymerase, the 45 and 62 access ory proteins, and 32protein are cross-l inked to the primer at specific positionsin the presence of ATP, but there is no detectable cross-l inking of the 44 access ory protein (Figure 4). In this andthe fol lowing figures the cross-l inked products were boi ledand analyzed on a 12% SDS-polyacrylamide gel. Mos t ofthe dark band at the top of the gels is from a small fractionof pr imer tha t reanneals to the template after boi ling of thesample, since i t is present at about the same intensity innonphotolyzed controls. The positions of the free proteinsare shown on the left of each panel and those of the ad-ducts on the r ight. The cr i ter ia used for the identi f icationof each adduct are described in the Experimental Proce-dures.

The efficient cross-l inking of polymerase (943 protein)at position -4 is dependent on ATP, the three ac cessoryproteins, and 32 protein (Figure 4A, bottom, lane 2 versuslanes 5 and 7-9). The low level of polymerase cross-l inkedto the primer in the absence of ATP or any of the otherproteins (Figure 4A) is the same as that observed withpolymerase alone (data not shown) and is much less thanthat observed with polymerase alone with the 1 /20-mer(see Figure 2) because polymerase can also bind to thelong single-stranded @Xl 74 template. Thus , on the longertemplate an ATP-dependent reaction involving the acces-sory proteins and 32 protein is necessary to preferential lybind polymerase to the primer terminus. In the presenceof the access ory proteins, polymerase is cross-l inked tothe primer at position -4 (Figure 4A, lane 2) but not -9( lane 3) or -14 ( lane 4), consistent with cross-linking ofpolymerase alone at pr imer positions -2 to -5 with the 1 I/20-mer (see Figure 2).

Cross-l inking of the 45 access ory protein is mos t effi-cient at positions -14 and -20. This cross-l inking requiresATP and the 44/62 and 32 proteins, and is decreased inthe absence of the polymerase. In reactions with the 34(4*)primer-tem plate and all five replication proteins , 45 proteinis cross-l inked at position -14 (Figure 4A, top, lane 4) butnot at position -4 (lane 2) or -9 (lane 3). 45 Protein canalso be cross-l inked from -20 to -30 using the 34(20*)primer in which the cross-linka ble residue is initially a t -20(Figure 4C ). The cross-l inking is stronger at -20 ( lane 2)than at -25 (lane 3) or -30 (lane 4). The m obility of the45 protein-DNA adducts dec reases as the 34(20*)-mer iselongated to the 39(25*)-me r and 44(30)-mer (lanes 2-4). The experiments with the 34(4) pr imer w ere performedwith ATP contaminated with small amounts of dATP, re-sulting in production of some 47-mer in addition to theexpected 44-mer (see Figure 3A, lane 3). Thus, i t was


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Figure 4. Cross-Linking the T4 DNA Repl icat ion Proteins in the Presence of ATPUnless otherwise indicated, react ions contained T4 DNA polymerase, 44162 and 45 accessory proteins, 32 protein, a nd ATP. The posi t ions of theDNA-protein adducts are shown on the right and those of the f ree protein standards on the lef t of each g el .(A) Cross-l inking of proteins to bases 4,9, or 14 f rom the primer terminus. The autoradiogram of a 12% SDS-PAGE gel shows proteins cross-l inkedto the 5 -[ P]34(4)-mer (dTTP, lanes 2 and 5-9). 39(9)-mer (dTTP and dCTP, lane 3) and 44(14)-mer (dTTP, dCTP, and dGTP, lane 4).Cross-l inking to polymerase (943 product) is shown in the shorter exposure at the bot tom, and cross-linking to the 32 ssDNA-binding protein andthe 45 and 62 accessory proteins in the longer exposure is shown at the top.(B) Cross-l inking of T4 45 accessory protein 14 bases f rom the 3 primer term inus requires ATP. 44/62 accessory protein complex, and 32ssDNA-binding protein. The 34(4)-mer (dlTP, lane 2) was elongated to the 39(9)-mer (dTTP and dCTP, lane 3) or the 44(14)-mer (dTTP, dCTP,and dGTP, lanes 4-6).(C) Cross-l inking of T4 DNA repl icat ion proteins to bases 20, 25, and 30 f rom the 3 primer terminus. The 34(20)-mer (dTTP, lanes 2 and 5-10)was elongated to the 39(25)-mer (dlTP and dCTP, lane 3) or the 44(30)-mer (dlTP, dCTP, and dGTP, lane 4). Note that 45 protein cross-linkingto the 34(20)-mer is decreased in the absence of DNA polymerase (943) and that 62 protein cross-linking is increased in the absence of ATP orthe 45 protein. There is no protein cross-linking in the presence of DTT, which destroys the cross-linkable azido moiety (lane 10).

possible that the labeled 45 protein seen in Figures 4A and48 resulted from cross-l inking only to the 47(17*)-mer.When the cross-l inking reaction wa s performed with theHPLC-puri f ied ATP, the amount of the 47-mer producedwas reduced (see Figure 3A, lane 6), but the intensity ofthe 45 protein cross-l inking was not detectably changed(data not shown), consistent with cross-linking of the 45protein to both the 44(14*)-mer and 47(17*)-mer. The re-quirements for cross-l inking the 45 protein at -14 and -20are shown in Figures 48 and 4C, respectively. There isno detectable cross-l inked 45 protein when ATP or 44/62complex is excluded ( lanes 5 and 6). Only a trace of cross-l inked 45 protein is evident in the absence of the 32 protein(Figure 48, lane 8; Figure 4C, lane 7). Cross-l inking of45 protein at -20 is greatly decreased in the absence ofpolymerase (Figure 4C, lane 9).

The 62 protein component of the 44162 access ory pro-tein complex is only weakly cross-l inked to the primer inthe presence of the five repl ication proteins and ATP, andthere is no detectable cross-l inking of 44 protein to theprimer (Figure 4A, top). The weak cross-l inking of the 62

protein requires 32 protein (lane 9), but is observe d in theabsence of ATP, or the polymerase or 45 proteins ( lanes5, 6, and 6 ). Thus, i t is not clear that the 62 protein cross-l inked in the presence of the five repl ication proteins isactual ly on a primer with polymerase and 45 protein.Cross-l inking of 62 protein at -4 increases in the absenceof polymerase (Figure 4A, lane 6) suggesting that i t isbinding to a si te sequestered by polymerase in the com-plete reaction . Similarly, 62 protein cross-linking at -20 isobserved only in the absence of ATP or the 45 protein(Figure 4C, lanes 5 and 8).

32 Protein is cross-l inked to the 34(4*) and 34(20*)primer under al l conditions (Figures 4A-4C), and can becross-l inked in the absence of any other proteins (data notshown). We cannot el iminate the possibi l i ty that some ofthis cross-l inking is to free single-stranded primer, al-though little free primer is presen t after isolation of theprimer-tem plate by gel filtration, and residual free primerwould be rapidly degraded by the exonuclease activi ty ofthe polymerase that show s a strong preference for single-stranded DN A. 32 Protein cross-l inking at -4 is increased


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1 0 3 0 5 0 0 3 0 50 3 0 5 0 0 3 0 5 02 0 ,O 6 0 2 0 4 0 6 0 0 4 0 6 2 0 4 0 6 0 J:

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Figure 5. ATP7S Inhibits Association of T4 DNA Polymerase and theAccessory Proteins on a Primed Single-S tranded Template but DoesNot Decrease the Rate of Synthesis by Polymerase and the AccessoryProteins Previously Bound to the PrimerThe primer-template (1.6 nM) was M13mp 2 DNA annealed to a syn-thetic 64-mer complementary to positions 6196 to 6261 of Ml 3mpl9.The 3 34 bases are complementary to M13mp2 , leaving a 50 baseunpaired tail. The reaction components were otherwise the same asthose used in the cross-linking reactions except tha t polymerase waspresent at 0.2 nM. 32 protein at 974 nM, dATP, dGTP, and dTTP at250 pM, and lPP]dCT P (600 cpmlpmol) at 125 uM. Reaction mixtureswere incubated for 1 min at 24OC before the addition of polymerase.Where indicated, ATpYS (1 mM) was added either before the acces-sory proteins or 10 s after the polymerase. DNA synthesis was stoppedby adding 3.5 pl aliquots to 2.5 ul of 0.2 M EDTA. and the productswereanalyzed on a 0.6% alkaline agarose gel as described (Venkatesan etal., 1962).

in the absence of ATP, polymerase, or the access ory pro-teins (Figure 4A, see also Figure 6A). It is not clear w hetherthis 32 protein cross-linking requires trans ient unwindingof the 3 region of the prim er.

Taken together, these cross-l inking studies with the34(4*) and 34(20) pr imers and ATP have al lowed us todetermine the position of the polymerase and 45 proteinsin the five protein sys tem . Since cross-l inking of polymer-ase and 45 protein depends on al l f ive proteins and ATP,32 protein is clearly required for efficient assem bly of thepolymerase-acces sory protein complex.ATP Hydrolysis Is Required to Cross-LinkT4 Polymerase to the PrimerATP hydrolysis is essential for the stimulation of T4 DNApolymerase by i ts access ory proteins, since ATPT S, whichcannot be hydrolyzed by the 44162 protein ATPas e, inhib-i ts stimulation of DNA synthesis by the access ory proteins(Piperno and Alberts, 1976). Kinetic studies with ATPySusing primed Ml3 DNA suggested that this analog doesnot decrease the rate of synthes is by previously formedpolymerase-acces sory protein complex es, but does pre-vent further association of polymerase with the access oryproteins (Jarvis et al ., 1990). How ever, in these studies therate of synthes is was l imited by a very low ratio of 32protein to DN A. Figure 5 show s that ATPyS has the sameeffect at the DNA, access ory protein, and 32 protein con-

centrations used in our cross-l inking studies, w here T4polymerase can completely copy a 7.2 kb Ml3 DN A tem-plate in 60 s at 24%. In this experiment the polymeraseconcentration was decreased to 0.2 nM so that i t would bemuch lower than that of the primer-template (1.6 nM) inorder to decreas e the probability of rebinding to previous lyelongated primers. At this low polymerase concentrationDNA synthes is was not detectable in the absence of theaccess ory proteins, or when ATPyS was added before theaccess ory proteins (Figure 5). If addition of ATPyS wasdelayed unti l 10 s, the size and intensity of the longestproducts present at each time point were the same asthose observed in the absence of the analog. There werefewer shorter products with ATPT S, co nsistent with theelongation of new chains beginning after 10 s when ATPySwas omi t ted.

To define the role of ATP hydrolysis in the assem bly ofthe polymerase-acces sory protein complex on the primer,we have compared the cross-l inking of these proteins tothe primer in the presence of ATP and ATPTS. We findthat replacing ATP with ATPTS changes the position andintensity of the cross-l inking of each o f the three access oryproteins, and prevents access ory protein-dependent cross-l inking of polymerase to the primer. The proteins cross-l inked at positions -4 to -14 in the presence of ATP orATPyS are compared in Figure 6A and those at -20 to -30in Figure 68. The specific radioactivi ty of the 34(4*)-merused in Figure 6A was the s ame as that of the 34(20*)-merused in Figure 66 so that the intensity of the bands in thetwo gels can be directly compared. The l ighter exposureat the bottom of Figure 6A indicates that, in contrast to thestrong cross-l inking of polymerase at -4 with ATP (lane 3)cross-l inking to polymerase with ATPyS is at the same lowlevel observed without ATP ( lanes 10 and 2) and does notdepend on the accessory proteins ( lane 10; lanes 14 and15). The heavier exposure at the top of Figure 6A show sfirst that a new species is apparent with ATPyS whose gelmobil i ty is consistent with cross-l inked 44 protein ( lanes10-13). 44 Protein is cross-link ed at positions -4 to -14(Figure 6A, lanes lo-12), but it is not cross-l inked at posi-tions -20 to -30 (Figure 6B, lanes 10-12). Cros s-linkingof 44 protein occurs in the absence of polymerase (Figure6A, lane 13), but not in the absence of the 45 or 32 proteins(Figure 6A, lanes 15 and 16 ).

The cross-linking of 62 protein is far greater in the pres-ence of ATPyS than in the presence of ATP (Figure 6A,top). The 62 protein adduct increases in intensity from-4 to -14 and its gel mobil i ty decreases as the primer iselongated (Figure 6A, lanes 1 O-l 2). It is barely detecta bleat -20 (Figure 6B, lane 10). Like 44 protein, 62 protein iscross-l inked in the absence of polymerase (Figure 6A, lane13) but there is little or no cross-link ing witho ut the 45 or32 proteins ( lanes 15 and 16). Figure 7 compares cross-l inking of the 44 and 62 proteins at -4 in the absence ofpolymerase without ATP ( lanes l-3), w ith ATPyS ( lanes4-6) and with ATP ( lanes 7-9). 45 Protein has no effecton the low level of cross-l inking of 62 protein without ATP(lane 3) but it increas es cross-link ing of 62 protein to theprimer with ATPy S (lane 6) or ATP (lane 9), and is requiredfor cross-l inking of 44 protein with ATPyS ( lane 6).


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Figure 6. Replacing ATP with ATPyS Changes the Pattern of Cross-Linking of the T4 Repl icat ion ProteinsUnless otherwise indicated, react ions contained T4 DNA polymerase, the 44162 and 45 accessory proteins, and 32 protein.(A) Cross-l inking of proteins to bases 4 to 14 f rom the 3 primer terminus. The 34(4)-mei, present in assays wi th only dTTP, was elonga ted to the39(9)-mer wi th dTTP and dCTP or the 44(14)-mer wi th dTTP, dCTP, and dGTP. The shorter exposure at the bot tom shows that in contrast torelat ively st rong cross-l inking of polymerase (943) at --4 in the presence of ATP (lane 3) wi th ATPyS (lane IO), cross-linking of polymerase doesnot increase above the level seen in the absence of ATP (lane 2) or !he accessory proteins (lanes 7, 6, 14, and 15). The longer exposure at thetop demonstrates that the 44 accessory protein is cross-l inked in the presence of ATPrS but not ATP and that ATPyS increases the cross-linkingto 62 accessory prote in, but abolishe s cross-linking of 45 accessory prote in to the 44(14)-m er. Note that cross-linking to the 44 and 62 proteinsrequires the 45 and 32 proteins (lanes 15 and 16) out occurs in the absence of polymerase (943) (lane 13).(B) Cross-l inking of 45 protein to the 34(20)-mer in the presence of ATPyS requires the 44/62 protein complex and 32 protein, but not polymerase.The 34(20)-mer, present in assays wi th on!y dlTP, was elongated to the 39(25)-mer wi th dTTP and dCTP or to the 44(30)-mer wi th dTTP, d CTP,and dGTP. Lane 17, showing cross-linking of 44 protein to the 34(4)-mer in the presence of ATPyS, is ident ical to lane 10 in (A) and is includedfor comparison.

The cross-l inking of 45 protein also changes whenATP-,S replaces ATP. 45 Protein is no longer cross-l inkedat -14 (see Figure 6A, lane 12 vers us lane 5). 45 Proteinis labeled at position -20, but the labeling is less intensethan in the presence of ATP (see F igure 6B, lane 10 versuslane 3). With ATP$S, cross-l inking of 45 protein at -20 isstill dependent on the 44/62 and 32 proteins (Figure 6B,lanes 14 and 16) but is no longer dependent on polymerase(lane 13j.

in summ ary, the cross-l inking studies in the presence ofATPyS demonstrate the assem bly of an access ory pro-tein-DNA complex that does not include or depend onpolymerase. Moreover, with the nonhydrolyzable ATP an-alog, the position and intensity of the cross-l inking of eachof the three accessory proteins differ from those found withATP.DiscussionProcessive DNA synthes is by the bacteriophage T4 pro-teins occurs on 32 protein-covered DNA templates, and

I

requires ATP hydrolysis, polymerase, the 44/62 and 45polymerase access ory proteins, as wel l as a primase-hel icase (Nossal and Alberts, 1983). Interaction of the T4proteins can be readi ly demonstrated by the effect of oneprotein, or set of proteins, on the reaction catalyzed byanother (for example see Richardson et al., 1990) but thephysical interactions between the proteins are too weakto al low isolation of the multienzyme complex es. Foot-printing techniques, which have been so informative withsequence-specific DNA-binding proteins, have been lesshelpful with repl ication proteins that must function on al lDNA sequences and move on the DN A during incorpora-tion and excision.

To establ ish how and where T4 DNA polymerase and i tsthree access ory proteins assemble on the primer-templatefor processive synthes is, we have used a cross-l inkingtechnique sensitive enough to detect weak protein-DNAinteractions. Using an 1 /20-mer containing a primer witha thymidine analog attached to a photoactivatable arylazide, we found that the polymerase alone interactsstrongly with the fi rst 5-7 bases of the primer. Similar re-


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,AAmbly of the T4 Repl icat ion complex

-g32-g44

-g62

g45-

Figure 7. Cross-Linking of the 44 and 62 Polymerase Accessory Pro-teins in the Absence of DNA PolymeraseReact ions contained the 34(4*)-8X174 hybrid, dTTP, 32 protein, andaddi t ional proteins as indicated. 45 Protein is required to cross-l ink 44protein in the presence of ATPyS and increases cross-linking of 62protein to the 34(4)-mer in the presence of ATP or ATPyS.

suits were obtained previously with the Klenow fragm entof Escherichia col i pol I (Catalan0 et al., 1990). For thestudy of T4 polymerase and i ts access ory proteins, weused two 34mers of identical sequence complem entaryto a @X174 template that differed only in the placementof the cross-l inkable residue, which was either 4 or 20residues from the primer terminus. The template se-quence al lowed the polymerase to extend the primer insteps of 5 bases with different dNTP com binations. Thus,we were able to examine the interaction of the repl icationproteins at 5 base intervals over a 30 base region of theprimer. Since assembly of the proteins and primer exten-sion is fast relative to cross-l inking, i t is l ikely that we arecross-link ing those proteins involved in incorporation andexcision of dNM P a t the primer terminus. Using thisprimer-template, we have shown that the polymerase andaccess ory proteins assemble into different complexes inthe presence of ATP and ATPyS and have mappedprimer-protein contac ts in each comp lex. In interpretingour results i t is important to note that detection of protein-DNA contacts with this technique requires that the nitrenegenerated upon photolysis be adjacent to an amino ac idcapableof forming a covalent adduct and that the resultantadduct be stable under our conditions. Since neither the

reactivi ty of the different amino acids with nitrenes nor thestabi l i ty of their resultant adducts is wel l characterized(Bayley, 1983) the fai lure to detect protein-DNA contactsdoes not prove their absence. Final ly, we have assume dthat the DNA-protein complexesde tected by cross-l inkingare the majori ty species under each condition.The protein-primer contac ts w e have detected have al-lowed us to construct a model for the assem bly of T4 DN Apolymerase, i ts access ory proteins, and 32 protein on theprimer strand (Figure 6). In the presence of ATP, the 44/62and 45 proteins bind the 32 protein-covered primer-template. Cross-l inking studies with ATPTS indicate thatbefore ATP hydrolysis the 44/62 protein complex can becross-l inked by nucleotides -4 to -14 from the primer ter-minus, with 44 protein cross-l inked mos t strongly at -4and 62 protein mo st strongly at -14. The 45 protein isweakly bound at -20 (Figure 6A). With ATPyS the cross-l inking of each of the access ory proteins depends on theother acce ssory proteins and 32 protein, but not on DN Apolymerase, and cross-l inking to DN A polymerase doesnot increase above the low level seen in the absence ofATP. We propose that ATP hydrolysis by the 44 proteinresults in a conformational change in these proteins suchthat the 44 protein is no longer bound near th e primerand binding of the 62 protein is decreas ed (Figure 88).

B (ATP) m

1C (ATP+Polymerase)

!ioisio 14 9 4Figure 6. Mode l for the Assembly of the Polymerase Accessory Pro-teins and Polymerase at a Primer Terminus Based o n Cross-LinkingStudies(A) The ini t ial event is binding of the polymerase accessory proteinsto a 32 protein-covered primer-template in the presence of ATP, butbefore ATP hydrolysis. 44 Protein is bound f rom -4 to -14, moststrongly at the -4 posi t ion. 62 Protein is bound f rom -4 to -14. moststrongly at the -14 posi t ion. 45 Protein is bound at the -20 posi t ion.Before ATP hydrolysis there is no accessory protein-dependent bind-ing of the polymerase (43 protein).(B) Af ter ATP hydrolysis, 44 protein moves away f rom the primer; 62protein is st i l l present at -4 and weakly bound at -20.45 Protein is st i l lpresent at -20.(C) Mov emen t of the accessory proteins as a result of ATP hydrolysisal lows polymerase to bind at the primer terminus to generate a complexcapab le of processive DNA synthesis. Polyme rase covers 5-7 basesof the primer. 45 Protein moves forward to the -14 posi t ion, and bind-ing of the 62 protein is decreased at -4. The polymerase and 45proteins are shown closer to the primer than the 62 protein, sincecross-linking to 62 protein is weak a nd is increased at -4 in the absenceof polymerase and at -20 in the absence of 45 protein. As discussedin the text , our cross-linking studies do not rule out the possibi l i ty thatthe 44/62 complex is released af ter binding of polymerase.


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Cd l25 6

Movem ent of the 44/62 protein complex away from theprimer al lows polymerase to bind to the primer term inus,generating an assem bly of T4 proteins capable of pro-cessive sy nthesis (Figure 8C ). The polymerase covers thefirst 5-7 bases of duplex DN A, and 45 protein move s for-ward to a position 14 to 20 bases from the 3 end. Move-ment o f the proteins is concerted in the sense tha t highlevel binding of polymerase occurs only in the presence ofthe three accessory proteins and ATP, and movem ent of45 protein depends on both polymerase and the 44162protein complex . These studies provide the first evidencethat, in the complex of the access ory proteins and polymer-ase, 45 protein is in direct contact with the DN A. We haveplaced the 44/62 protein complex on the primer in thepresence of the 45 and polymerase proteins and ATP (Fig-ure 8C), since a low level of cross-l inking to 62 proteinis observed under these conditions. 62 Protein is shownbehind polym erase and 45 protein in the mode l (Figure8C), because cross-l inking of 62 protein increases at -4in the absence of polymerase and at -14 and -20 in theabsence of 45 protein. Howe ver, since the cross-l inking to62 protein is weak and is also observed in the absence ofpolymerase and 45 protein, these studies do not rule outthe possibi l i ty that the movement of the 45 and polymeraseproteins, which depends on ATP hydrolysis by the 44 pro-tein, leads to release of the 44/62 complex from the primerstrand or possibly from the whole primer-template.

It is clear from studies of the accessory protein stimula-tion of the exonuclease activi ty o f T4 polymerase that morethan 3 nucleotides of template strand ahead of the primerare required for this stimulation (Venkatesan and Nossa l,1982). Thus an attractive possibi l i ty is that the 44/62 com-plex mov es forward from the primer to the template strandas the polymerase is clamped for processive synthesis ordegradation. We are currently using bifunctional proteincross-l inking reagents and primer-templates with thecross-l inkable residues in the template strand to definefurther the protein-protein and protein-DNA contac ts ofthese T4 repl ication proteins.Our finding that ATP hydrolysis is required for the as-semblyo f a protein-primer complex that includes polymer-ase is in accord with kinetic studies with ATPyS showingthat this analog prevents further asso ciation of the poly-merase with the access ory proteins, but does not de-crease the rate of synthesis by previously formed com-plexes of the polymerase and the access ory proteins onsingle-stranded DNA templates (Figure 5 and Jarvis et al .,1990). Alberts et al . (1980) have shown that ATPyS inhibitssynthes is on duplex templates more rapidly than synthes ison single-stranded templates, and have proposed that fre-quent partial reassembly of the T4 access ory protein-poly-merase com plex is required during strand displacementsynthes is. Our cross-l inking results are also consistentwith footprinting experiments (Munn, 1986; Sel ick et al .,1987) showing that with ATPyS the access ory proteinsprotect about 20 bases of the primer-template. The acces-sory proteins did not give a strong footprint to the primer-template with ATP, presumably because of the proteinmovement that accompanies ATP hydro lys is.E. col i DN A pol I l l and human polymerase 6 also require

ATP hydrolysis by polymerase access ory proteins to se-cure the polymerase to the primer-template for processivesynthes is. E. col i pol I l l works in conjunction with themultimeric y comp lex, s protein, and SSB (Wickner, 1976;ODonnell , 1987; Maki and Kornberg, 1988; McH enry,1988); human pol 6 uses multimeric RF -C (or Al), PCN A,and the RF-A ssDNA-binding protein (Tsurimoto andSti l lman, 1989; Wold et al ., 1989; Lee and Hurwitz, 1990).Theisolatedy6subunitsofthe E. col iycomplex(ODonnelland Studwell , 1990) and human RF-C (Tsurimoto andSti l lman, 1990) have DNA-dependent ATPase activi t iesthat may be analogous to that of the T4 44162 proteincomplex. The RF-C ATPase is st imula ted by PCNA, as the44/62 protein is stimulated by 45 protein.

The position-specific cross-l inking technique describedin this paper can define weak DNA-protein interactionsand identi fy which compone nts of multienzym e sys tem sare in direct conta ct with the DN A. It should prove usefulin determining the assem bly pathwa ys, structure, and pro-tein-DNA move ments in other multienzym e repl ication,recombination, and transcription sys tem s.Experimental ProceduresEnzymesThe T4 polymerase used for cross-linking experiments wi th the 1 I I20-mer was expressed f rom the plasmid pTL43W, a generous gi f f ofW. H. Konigsberg (Lin et al . , 1967) and puri f ied as described by Rushand Konigsberg (1989). For al l other experiments, T4 DNA polymerase,gene 45 and 44/62 polymerase accessory proteins, and gene 32single-stranded DNA-binding protein were puri f ied f rom T4 infectedcel ls as described by Venkatesan and Nossal (1982).

DNAsThe 1 /20-mer was prepared as previously described (Catalan0 et al . ,1990). The 34-mers were synthesized at the Penn State BiotechnologyInst i tute wi th a Mi l l igen Mode l 7500 DNA Synthesizer using phosphi ty-lated 5-(f luoroenylmethoxycarbonyl-3-aminopropyl)-2Ldeoxyuridine atthe desired posi t ion and puri f ied by reverse-phase HPLC as describedby Gibson and Benkovic (1987). The ol igomers were further puri f iedon a denaturing (8 M urea) 20% polyacrylamide gel and extracted wi than Elutrap (Schleicher and Schuel l ) in TBE buf fer. The detritylated,puri f ied 34-mers were derivat ized wi th N-5-azido-2+i trobenzoyloxy-succinimide, puri f ied by reverse-phase HPLC, and 5end-labeled wi th[yJ2P]ATP as described by Gibson and Benkovic (1987). Thiols, suchas di thiothrei tol (DTT) or mercaptoethanol , which destroy the azide,were not added to the kinase react ion. The 34-mer was annealed toQXDNA (primer: template, 2:1), and the hybrid was isolated by f i l t rat ionon a 7 ml co lumn o f Sepharose C l -2B in 20 mM ammonium acetate ,50 PM EDTA.

Nucleot idesNonradioact ive nucleot ides were f rom Pharmacia (Ul t rapure), exceptATPvS (Boehringer Mannhe im). [Y-~P]ATP was f rom DuPont New En-gland Nuclear.HPLC Puri f icat ion of rATPrATP was puri f ied f ree of dATP o n a Waters Del ta-Pak Cl8 (3.9 mm x30cm)column usingcondi t ionssimi larto thosedescribed bystocchietal . (1985). rATP was observed to elute at 7-8 min and dAT P at 12-14min. The f ract ions containing rATP were di luted P-fold wi th water; theresul tant solut ion was t i trated to pH 7.5 wi th KOH a nd the phosphatewas removed by adsorbing the ATP on a 2 ml column of DEAE A-25and e lut ing wi th a gradient of 20 ml each of 50 mM and 1 M triethylam-monium bicarbonate (pH 7.6).Repl icat ion React ions and Cross-Linking of Proteins to DNAUnless otherwise indicated in the f igures, a mixture of the five repl ica-t ion proteins was added to a mixture of the primer-template and nucleo-


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gmbly of the T4 Repl icat ion Complex

t ides (at room temperature) in a 0.5 ml polypropylene microcentri fugetube to give f inal concentrat ions of 25 PM ATP or AT-S, 20 pM eachdNTP, 25 mM Tris acetate (pH 7.5), 60 mM potassium acetate, 6 mMmagnesium acetate, 0 .2 mglm l bovine serum albumin, 2 nM (approxi-mately 2 x IO cpmlpmol) 5 -rP]34(4*) or 34(20)mer-0X174 DNAduplex, 1.5 pg/ml(l4.6 nM) T4 DNA polymerase, 4 ug/ml44/62 proteincomplex, 6 kg/ml 45 protein, and 26 wg/ml(614 nM) 32 protein in a totalvolume of 20 ~1. The molar concentrat ions of the accessory proteinscannot be given since they are mult imeric, and there is disagreementabout the number of subuni ts in each mult imer (Jarvis et al . , 1989a;Rush et al . , 1969). DlT was not added to the react ions, but there wasresidual DTT (


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funct ional analysis of the bacteriophage T4 DNA repl icat ion complex.Molecular m echanisms in DNA repl icat ion and recombinat ion. UCLASymp. Mol. Cel l . Biol . 127, 261-275.Lee, S.-H., and Hurwitz, J. (1990). Mecha nism of elongat ion of primedDNA by DNA polymerase 5, prol i ferat ing cel l nuclear ant igen and act i -vator 1. Proc. N at l . Acad. Sci . USA 87, 56725676.Lin, T.-C., Rush, J. , Spicer, E. S. , and Konigsberg, W. H. (1987). Clon-ing and expression of T4 polymerase. Proc. Nat l . Acad. Sci . USA 84,7000-7004.Mace, D. C. , and Albert% B. M. (1984). The com plex of T4 bacterio-phage g ene 44 and 62 repl icat ion proteins forms an ATPase that isst imulated by DNA an d by T4 gene 45 protein. J. Mol . Biol . 777, 279-293.Maki , S. , and Kornberg, A. (1988). DNA polymerase I l l holoenzyme ofEscherichia co/ i . I I . A novel complex including the y subuni t essent ialfor processive synthesis. J. Biol . Chem. 263, 6555-6560.f&Henry, C. (1988). DNA polymerase I l l of Eschericbia co/ i . Annu.Rev. Biochem. 57, 519-550.Munn, M. (1986). Analysis of the bacteriophage T4 DNA repl icat ioncomplex. PhD thesis, Universi ty of Cal i fornia, San Francisco, Cal i -fornia.Nossal , N. G., and Alberts, B. M. (1983). The mechanism of DNArepl icat ion catalyzed by puri f ied T4 DNA repl icat ion proteins. In Bacte-riophage T4, C. K. Mathews, E. M. Kut ter, G. Mosig, and P. B. Berget ,eds. (Washington, DC: American Society for Microbiology), pp. 71-81.ODonnel l , M. E. (1987). Accessory proteins bind a primed templateand mediate rapid cycl ing of DNA polymerase I l l holoenzyme fromEscbericbia co/ i . J. Biol . Chem. 282, 16558-16565.ODonnel l , M. E. , and Studwe l l , P. (1990). Total reconst i tut ion of DNApolymerase I l l holoenzyme reveals dual accessory protein clamps. J.Biol . Chem. 265 , 1179-1187.Piperno, J. Ft . , and Alberts, B. M. (1978). An ATP st imulat ion of the T4DNA polymerase mediated via T4 gene 44/62 and 45 proteins. Therequirement for ATP hydrolysis. J. Biol . Chem. 253, 5174-5179.Piperno, J. R. , Kal len, R. G., and Alberts, B. M (1978). Analysis of aT4 DNA repl icat ion protein complex. Studies of the DNA recogni t ionsi te forT4 gene 44/62 and 45 protein-catalyzed ATP hydrolysis. J. Biol .Chem. 253, 5180-5185.Richardson, R. W., and Nossal , N. G. (1989a). Characterization of thebacteriophage T4 gene 41 DNA hel icase. J. Biol . Chem. 254,4725-4731.Richardson, R. W., and Nossal , N. G. (1989b). Trypsin cleavage in theCOOH terminus of the bacteriophage T4 gene 41 DN 4 hel icase al tersthe primase-hel icase act ivi ties of the T4 repl icat ion complex in vi t ro.J. Biol . Chem. 254, 4732-4739.Richardson, R. W., El l is, R. L. , and Nossal , N. G. (1990). Protein-protein interact ions wi thin the bacteriophage T4 DNA repl icat ion com-plex. Molecular mechanisms in DNA repl icat ion and recombinat ion.UCLA Sym p. Mol. Cel l . Biol . 727, 247-259.Rush, J. , and Konigsberg, W. H. (1989). Rapid puri f icat ion of overex-pressed T4 DNA polymerase. Prep. Biochem. 19, 329-340.Rush, J. . Lin, T.-C., Quinones, M., Spicer, E. K. . Douglas, I . , Wi l l iams,K. R. , and Konigsberg, W. H. (1989). The 44P subuni t of the T4 poly-merase accessory prote in comp lex catalyzes ATP hydrolysis. J. Bio l.Chem. 264, 10943-10953.Sel ick, H. E. . Barry, J. , Cha, T.-A. , Munn, M., Nakanishi . M.. Wong, M.L. , and Alberts, 8. M. (1987). Studies on the T4 bacteriophage DNArepl icat ion system. DNA repl icat ion and recombinat ion. UCLA Symp.Mol. Cel l . Biol . 47, 183-214.Stocchi , V. , Cucchiarini , L. , Magn ini , M ., Chiarant ini , L. , Palma, P. ,and Crescent ini, G. (1985). Simultaneou s extract ion and reverse-phase high performance l iquid chromatographic determinat ion of ade-nine and pyridine nucleot ides in human red blood cel ls. Anal . Biochem.146, 118-124.Tsurimoto. T. . and St i l lman , 8. (1989). Mul t iple repl icat ion factors aug-ment DNA synthesis by the two eukaryotic DNA polymerases, a and6. EMBO J. 8, 38833889.Tsurimoto, T. , and St i l lman . B. (1990). Funct ions of repl icat ion factor

C and prol i ferat ing-cel l nuclear ant igen: funct ional simi lari ty of DNApolymerase accessory proteins f rom human cel ls and bacteriophageT4. Proc. Nat l . Acad. Sci . USA 87. 1023-1027.Venkatesan, M., and Nossal , N. G. (1982). Bacteriophage T4 gene 44/62 and gene 45 polymerase accessory proteins st imulate hydrolysisof duplex DNA by T4 DNA polymerase. J. Biol . Chem. 257, 12435-12443.Venkatesan. M., Si lver, L. L. , and Nossal, N. G. (1982). BacteriophageT4 gene 41 protein, required for the synthesis of RNA primers, is alsoa DNA hel icase. J. Biol . Chem. 257, 12426-124 34.Wickner, S. (1976). Mechanism of DNA elongat ion by Escherichia co/ iDNA polymerase I l l , dna 2 protein, and elongat ion factors I and I I I .Proc. Nat l . Acad. Sci . USA 73, 3511-3515.Wold, M. S. , Weinberg, D. H. , Virshup, D. M., Li , J. J. , and Kel ly, T.J. (1989). Ident i f icat ion of cel lular proteins required for simian vi rus 40DNA repl icat ion. J. Biol . Chem. 264, 2801-2809.

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Assemby of DNA Replication Proteins_Todd L Capson