And Stimulation of DNA Polymerases a and 6 (DNA Unwinding/Proliferating-Cell Nuclear Antigen/Simian Virus 40 Large Tumor Antigen) MARK K

Proc. Natl. Acad. Sci. USA Vol. 86, pp. 9757-9761, December 1989 Biochemistry Multiple functions of human single-stranded-DNA binding protein in simian virus 40 DNA replication: Single-strand stabilization and stimulation of DNA polymerases a and 6 (DNA unwinding/proliferating-cell nuclear antigen/simian virus 40 large tumor antigen) MARK K. KENNY, SUK-HEE LEE, AND JERARD HURWITZ Graduate Program in Molecular Biology, Sloan-Kettering Cancer Center, New York, NY 10021 Contributed b) Jerard Hurwitz, September 20, 1989

ABSTRACT The human single-stranded-DNA binding bly related to replication factor C) results in a marked protein (human SSB) is required for simian virus 40 (SV40) increase in DNA synthesis and, in particular, the length ofthe DNA replication in vitro. SV40 large tumor antigen and human DNA products. It is possible that in this dipolymerase SSB can support extensive unwinding of SV40 origin- replication pathway, pola may be the lagging-strand poly- containing DNA in the presence of ATP and a topoisomerase merase, and polS may be responsible for leading-strand that relieves positive superhelicity. Although SSBs from viral synthesis (12-15, 29, 30). RNase H, 5' -+ 3' exonuclease, and prokaryotic sources substituted for human SSB in the DNA ligase, and topo II can then remove RNA primers, seal DNA-unwinding reaction, they did not substitute in the repli- nicks, and decatenate the daughter molecules (10, 11). Stim- cation of SV40 DNA. The specificity for human SSB in SV40 ulatory and specificity factors and proteins required for DNA replication can be explained, at least in part, by the assembly and replication of chromatin are also likely to be finding that DNA polymerase a was stimulated 10-fold by involved in SV40 DNA replication (31-33). human SSB but not by other SSBs. Human SSB also stimulated Human SSB (also known as replication factor A, replication proliferating-cell nuclear antigen-dependent DNA polymerase protein A, and HeLa SSB) is a multisubunit protein containing 6; however, other SSBs stimulated this polymerase as well. polypeptides of 70, 34, and 11 kDa (6-8, 11). Here, we characterize the role of human SSB in SV40 DNA replication Replication ofsimian virus 40 (SV40) DNA resembles cellular in vitro. We present evidence that human SSB serves addi- DNA replication in many respects and provides an excellent tional functions in replication aside from its role in DNA model for the study of eukaryotic DNA replication (for unwinding. Human SSB markedly stimulated pola and pol3. reviews, see refs. 1-4). SV40 large tumor antigen (TAg) is the Although other viral and prokaryotic SSBs stimulated polI, only viral protein required for replication; the host provides the stimulation of pola was specific to human SSB. all other replication proteins. Presumably many of these host proteins are also involved in cellular DNA replication. Since the development of an efficient in vitro system for - MATERIALS AND METHODS replication of SV40 DNA (5), many enzymes have been Proteins, DNAs, and Other Reagents. The following re- identified that are thought to play a role in SV40 replication. agents were obtained commercially: unlabeled nucleotides These proteins include human single-stranded-DNA binding (Boehringer Mannheim), bacteriophage T4 gene 32 protein protein (SSB) (6-8), DNA polymerase a-primase complex (T4g32) and (dT)12_18 (Pharmacia), radionucleotides (New (pola-primase) (9), topoisomerases (topo) I and II (10), England Nuclear), (dA)4000 (Life Sciences, Saint Petersburg, RNase H, a 5' -> 3' exonuclease, DNA ligase (11), prolifer- FL), creatine phosphokinase (Worthington), and chloroquine ating-cell nuclear antigen (PCNA) (12), PCNA-dependent (Sterling-Winthrop Research Institute). Bovine serum albu- DNA polymerase 8 (pol) (13), and other partially charac- min (Miles) was heat-denatured prior to use. TAE buffer (1x) terized factors (14-16). is 40 mM Tris acetate, pH 8/1 mM EDTA. The role(s) of each of these proteins and the mechanisms The SV40 origin-containing plasmid pSV01AEP has been involved in SV40 DNA replication has begun to be eluci- the ammo- dated. An initial event in the replication pathway is the described (34). Preparation of 35-65% (wt/vol) ATP-dependent binding of 12 TAg molecules to the 64- nium sulfate fraction of HeLa crude extract (6), adenovirus base-pair core origin of replication, resulting in a bibbed DNA binding protein (Ad DBP) (35), topo I (11), immunopu- nucleoprotein structure (17-20). The binding of TAg causes rified TAg (20), immunopurified HeLa pola-primase (11), conformational changes ofthe origin DNA, including a region HeLa PCNA (28), PCNA-dependent HeLa polS (13), and the of melted DNA (21). In the presence of human SSB and topo 0.4 M double-stranded-DNA-cellulose elongation inhibitor/ I or II, TAg can act as a helicase and extensively unwind the activator I fraction (14) have been described. HeLa SSB was DNA (22-25). This unwinding reaction appears to be a purified by a modification of the procedure of Wobbe et al. required step in the replication of SV40 DNA based on DNA (6) that will be published elsewhere, Escherichia coli SSB, mutational analysis and pulse-chase experiments (26, 27). herpes simplex virus infected-cell polypeptide 8 (ICP8), and Pola-primase can initiate DNA synthesis and produce short calf unwinding protein 1 (UP1) were generously provided by DNA chains. However, in the presence of a protein inhibitor Kenneth Marians (Sloan-Kettering Cancer Center), Michael identified as poly(ADP-ribose) polymerase, the further elon- O'Donnell (Cornell University Graduate School of Medical gation ofDNA chains by pola is blocked by the binding ofthis elongation inhibitor to the ends of the DNA chains (28). Abbreviations: SSB, single-stranded-DNA binding protein; SV40, Addition of pol5, PCNA, and the activator I fraction (possi- simian virus 40; TAg, SV40 large tumor antigen; pola, DNA poly- merase a; pol8, PCNA-dependent DNA polymerase 8; pol III, E. coli DNA polymerase III; topo, topoisomerase; PCNA, proliferating-cell The publication costs of this article were defrayed in part by page charge nuclear antigen; Ad DBP, adenovirus DNA binding protein; T4g32, payment. This article must therefore be hereby marked "advertisement" bacteriophage T4 gene 32 protein; ICP8, herpes simplex virus in accordance with 18 U.S.C. §1734 solely to indicate this fact. infected-cell polypeptide 8; UP1, calf unwinding protein 1.

9757 Downloaded by guest on September 28, 2021 9758 Biochemistry: Kenny et al. Proc. Natl. Acad. Sci. USA 86 (1989) Sciences), and Bruce Alberts (University of California, San human SSB in SV40 DNA replication, various SSBs were Francisco), respectively. tested for their ability to substitute in reactions in which SV40 DNA Replication Assay. Reaction mixtures (30 pl), human SSB is involved. The SSBs examined include those containing 40 mM creatine phosphate [diTris salt (pH 7.7)], from E. coli, bacteriophage T4 (T4g32), adenovirus (Ad 7 mM MgCl2, 0.5 mM dithiothreitol, 4 mM ATP, 200,uM DBP), and herpes simplex virus (ICP8) (for reviews, see refs. UTP, 200 ,M GTP, 200 ,M CTP, 100 AtM dATP, 100 AM 37 and 38). Also tested was the calf thymus protein UP1, dGTP, 100 AM dCTP, 20 AM [3H]dTTP (300 cpm/pmol), 1 jig which was originally thought to be a bona fide eukaryotic of creatine phosphokinase, 180 ng of pSV01AEP DNA, 10 Ag SSB but has subsequently been shown to be a proteolytic of bovine serum albumin, 0.4 jig of TAg, 180 Ag of the fragment of a heterogeneous ribonucleoprotein (38-42). 35-65% ammonium sulfate fraction, and SSB as indicated, The various SSBs were assayed in the in vitro SV40 DNA were incubated for 60 min at 37TC, and acid-insoluble radio- replication reaction using a crude HeLa cell fraction devoid activity was determined. of human SSB (Fig. 1). Only human SSB supported high SV40 DNA Unwinding Assay. Reaction mixtures (30 Al) levels of DNA synthesis. The level of nucleotide incorpo- containing 40 mM creatine phosphate [diTris salt (pH 7.7)], 7 ration observed with the other SSBs was <2% of that mM MgCl2, 0.5 mM dithiothreitol, 4 mM ATP, 1 ,ug ofcreatine detected with human SSB. However, E. coli SSB, Ad DBP, phosphokinase, 180 ng of relaxed covalently closed circular and ICP8 did produce small amounts of DNA synthesis that pSV01AEP DNA (form I'), 5 ,g of bovine serum albumin, 0.4 were clearly (2- to 4-fold) above background. ,ug TAg, 750 units of topo I, and SSB, as indicated, were Various SSBs Work in the SV40 DNA-Unwinding Reaction. incubated for 60 min at 37°C. Reactions were stopped by the The various SSBs were tested for their ability to support addition of a solution (5 ,ul) containing 0.1 M EDTA, 2% unwinding of SV40 origin-containing DNA in conjunction (wt/vol) SDS, proteinase K (1 mg/ml), and glycogen (1 with TAg and topo I. Human and E. coli SSBs have been mg/ml). Samples were further incubated for 15 min at 37°C and observed to function in this assay (22-24, 26). Agarose gel then ethanol-precipitated and electrophoresed at 20 V for 12 hr electrophoresis of the reaction products and the quantitation through a 1.5% agarose gel in lx TAE buffer containing of unwound DNA (form U) are shown in Fig. 2. Human SSB chloroquine (1.5 jig/ml). Gels were soaked in 50 mM NaCl for supported unwinding of up to 70% of the substrate DNA 60 min and then in ethidium bromide (0.5 ,ug/ml) for 60 min molecules. At low concentrations of human SSB, form U before visualizing DNA with a UV transilluminator. DNA Polymerase Assay. Reaction mixtures (30 pul) con- A tained 40 mM creatine phosphate [diTriq salt (pH 7.7)], 7 mM SSB: None Human E coli ICPS Ad DEP UT14 1 MgCl2, 0.5 mM dithiothreitol, 4 mM ATP, 1 ,ug of creatine phosphokinase, 20 ,uM [3H]dTTP (400 cpm/pmol), 5 ,ug of bovine serum albumin, 0.1 ,ug (10 ,uM as nucleotide) of (dA)4Qw(dT)12.18 [20:1 (wt/wt)], and SSB, DNA polymerase, and accessory factors, as indicated. After incubation at 37°C, acid-insoluble radioactivity was determined. RESULTS Other SSBs Cannot Replace Human SSB in the SV40 DNA 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 2. 22 2324 25 26 2: Replication Reaction. To gain insight into the functions of 120 B 80 70 Human

_ 60 o AdDBP 0~~~~~~~~~~~~ ICP8 0 5C 0 O 4C a- ) - 0 E/ coli----° E 80 D 30

0) tL 2C 0 60 - lC o _7 I ,Upi 1__ i i T4g32, CL I 0 0.5 1.0 1.5 2.0 0L SSB Added 40 - (;,g) 00 FIG. 2. SV40 DNA unwinding using various SSBs. Unwinding reaction mixtures contained SV40 origin-containing DNA, TAg, 20 HeLa topo I, and an SSB as indicated. (A) Reaction products were electrophoresed through a 1.5% agarose gel containing chloroquine (1.5 ,g/ml). The reactions contained the following amounts of the 0.05 and 0 t indicated SSBs. Lanes: 1, 0,ug; 2, 8, 14, and 20, ,4g; 3, 9, 15, 0 0.2 0.4 0.6 0.8 1.6 21, 0.1 ,ug; 4, 10, 16, and 22, 0.2 ,ug; 5, 11, 17, and 23, 0.5 ,ug; 6, 12, SSB(9g) 18, 24, 26, and 27, 1.0 ,g; 7, 13, 19, and 25, 2.0 ,ug. (B) Reaction products were quantitated by scanning a negative photograph of the FIG. 1. SV40 DNA replication using various SSBs. Replication gel with a laser densitometer (LKB 2202 Ultroscan). The percent of reactions contained SV40 origin-containing DNA, TAg, the 35-65% DNA molecules unwound in a given reaction was calculated as ammonium sulfate fraction of HeLa crude extract, and an SSB as follows: % form U = [form U/(form U + form I')] x 100. Form ll indicated: human SSB (a), E. coli SSB (o), ICP8 or Ad DBP (o), and (nicked circular duplex DNA) was not included in the calculation T4g32 or UP1 (o). Reaction mixtures were incubated for 60 min at since it is not a substrate for the reaction. The SSBs used were human 37°C, after which time the amount ofacid-insoluble radioactivity was SSB (o), E. coli SSB (o), ICP8 (o), Ad DBP (o), and T4g32 or UP1 determined. (A)- Downloaded by guest on September 28, 2021 Biochemistry: Kenny et al. Proc. Natl. Acad. Sci. USA 86 (1989) 9759

migrated as a diffuse smear, but at high concentrations form 35 U was a sharp fast-migrating band. Ad DBP and ICP8 Human produced comparably high levels of form U. The amount of unwound product observed in the presence of E. coli SSB 30 /~~~~~~~~~~ was significant but peaked at about 45%. The migration of form E 25- U produced with E. coli SSB was diffuse, presumably E reflecting different extents to which the DNA molecules were 0. unwound. T4g32 and UP1 did not support unwinding, thus n 20 - explaining their inactivity in the DNA replication assay. The 0 differences between human I.. SSB and the other SSBs (E. coli, 0 15 Ad DBP, and ICP8) in the unwinding assay do not appear to be sufficient to account for the finding that only human SSB lo / can support SV40 DNA replication. Human SSB must have E. 10 e AdDBP A some function in addition to its role in DNA unwinding. Human SSB Stimulates Pola. Many SSBs have been shown 5 Z^/°~Fl ~~ E coli to stimulate their cognate -~~~~~~~~~T4g32A-a DNA polymerases (37, 38). The - effect of human SSB on pola was tested using (dA)Qw, 2 ICP8 (dT)12_18 as a template primer. Human SSB enhanced the rate 0 0.2 0.4 0.6 0.8 1.0 of DNA synthesis by pola %10-fold when assayed under SSB Added(t1g) conditions used for SV40 DNA replication (Fig. 3). The stimulation was FIG. 4. Effect of various SSBs on pola activity. Reaction mix- only 3- to 5-fold when reaction conditions of tures contained 0.1 jug of (dA)Qwy(dT)12.18 [20:1 (wt/wt)], 0.05 unit lower ionic strength were used. The stimulation by human of pola-primase, and an SSB, as indicated [human SSB (e), E. coli SSB, however, was not dependent on ATP and an ATP- SSB (o), ICP8 (i), Ad DBP ((a), T4g32 (A), and UP1 (A)]. Reaction regenerating system (data not shown). mixtures were incubated for 60 min at 370C. The effect ofother SSBs on pola activity was also examined (Fig. 4). Optimal levels of human SSB stimulated DNA syn- (Fig. 5). Pol8 activity in the presence ofSSB and the auxiliary thesis =10-fold. Higher levels (>2 ug) were inhibitory, al- factors was dependent on ATP (data not shown). though it is not known whether this is a specific effect of the The effect ofvarious SSBs on the activity ofpol8 was tested. human SSB or due to an inhibitor contaminating the SSB Human SSB, E. coli SSB, Ad DBP, and T4g32 all stimulated preparation (data not shown). E. coli SSB, T4g32, and ICP8 pol8 in the presence of PCNA and the elongation inhibitor/ did not stimulate pola and in fact inhibited nucleotide incor- activator I fraction (Fig. 6A). Conversely, all of these SSBs poration. Low levels of Ad DBP had a slight (<2-fold) stim- inhibited pol8 when the elongation inhibitor/activator I frac- ulatory effect on pola activity, whereas higher levels were tion was omitted (Fig. 6B). However, we have observed inhibitory. UP1 enhanced the activity of pola as has been limited stimulation (<4-fold) of pol8 by low levels ofthe SSBs shown (41). However, as demonstrated in Fig. 2, UP1 was not in the absence of the elongation inhibitor/activator I fraction active in the DNA unwinding assay. Thus, although many using some preparations ofpoly(dA)-oligo(dT) (A. Kwong and SSBs can function in DNA unwinding, the ability of human J.H., unpublished observations). The effect of SSBs on polb SSB to stimulate pola could contribute to the high degree of activity, like DNA unwinding, appears to be nonspecific. UP1 specificity for human SSB in SV40 DNA replication. distinguished itself from the bona fide SSBs in this assay by Various SSBs Stimulate Polb. Pol8 has been shown (13) to having little or no effect on pol8 activity irrespective of the play a role in SV40 DNA replication. The activity of pol8 in presence or absence of the elongation inhibitor/activator I this system is dependent on PCNA and the elongation fraction. Thus, while UP1 enhanced pola activity, only human inhibitor/activator I fraction. The effect of human SSB on SSB stimulated both pola and pol8. pol8 was assayed on (dA)Qwy(dT)12_18. Human SSB enhanced the rate of DNA synthesis by pol8 -10-fold in the presence of PCNA and the elongation inhibitor/activator I fraction DISCUSSION Human SSB shares many of the properties associated with 35 SSBs. It binds to single-stranded DNA in preference to double-stranded DNA and requires >1 M NaCl to be eluted 30 + Human SSB 12 E 25 *5 10 _ a E 20 cL -08 ISSB 0 - 8 15 0 C 0 HL6 0~ 2 C H 10_/ 4 2 - Human SSB - Human SSB -o 6 0 10 20 30 40 50 60 0 10 20 30 40 50 60 Time(min) Time(min) FIG. 3. Effect of human SSB on the rate of DNA synthesis by FIG. 5. Effect ofhuman SSB on the rate ofDNA synthesis by pol&. pola. Reaction mixtures contained 0.1 ,ug of (dA)0w-(dT)12.18 [20:1, Reaction mixtures contained 0.1 ug of (dA)Qw-(dT)12.18 [20:1 (wt/ (wt/wt)], 0.05 unit of pola-primase, and 1.25 Ag of human SSB (0) wt)], 0.1 unit of poI8, 0.2 )mg of PCNA, 1.5 tug of elongation inhibitor/ or no SSB (a), as indicated. Reaction mixtures were incubated at activator I fraction, and 0.5 tug of human SSB (e) or no SSB (0), as 37TC as indicated. indicated. Reaction mixtures were incubated at 370C as indicated. Downloaded by guest on September 28, 2021 9760 Biochemistry: Kenny et al. Proc. Natl. Acad. Sci. USA 86 (1989) Consistent with this, we have found that protein phosphatase 30 B E A Human 2A, calf alkaline intestinal phosphatase, and bacterial alka- 0: 25 - ;-A line phosphatase could each activate "adenovirus" TAg for 0. oCLTg3* the unwinding reaction, while "COS cell" and "baculovi- rus" TAg were active in the absence of phosphatase treat- -0 15 ment (P. Bullock, F. Dean, M.K.K., and J.H., unpublished

0 observations). 10 a - The ability of human SSB to stimulate pola has been 0~ LT4g32 F- described here. This property appears to be relatively spe- E.coli cific since the other SSBs tested did not enhance pola ~0 i 0oI J A activity. The one exception was UP1, which, as documented 0 0.5 1.0 1.5 2.0 2.5 3.5 0 0.5 1.0 1.5 2.0 2.5 3.5 elsewhere (41), stimulated DNA synthesis by pola. This SSB Added (/±g) effect is probably fortuitous since UP1 has been shown to be FIG. 6. Effect of various SSBs on pol8 activity. (A) Reaction a proteolytic fragment of a heterogeneous ribonucleoprotein mixtures contained 0.1 Ag of(dA)woo-(dT)12-18 [20:1 (wt/wt)], 0.1 unit (42). Other proteins have also been identified that stimulate of pol8, 0.2 A&g of PCNA, 1.5 tkg of elongation inhibitor/activator I pola in vitro on synthetic templates, but they have not been fraction, and an SSB, as indicated [human SSB (e), E. coli SSB (o), shown to stimulate pola in the context of replication (46-51). Ad DBP (o), T4g32 (A), and UP1 (A)]. Reaction mixtures were The specificity of the stimulation of pola by human SSB .ncubated for 60 min at 37TC. (B) Same as in A except the inhibitor/ suggests the likelihood of an interaction between pola and activator I fraction was omitted. human SSB. The effect of human SSB on pola was tested from single-stranded-DNA-cellulose (6-8). It stabilizes re- using (dA)4000 (dT)12-18 at a 20:1 ratio. Human SSB was also gions of single-stranded DNA, and it stimulates the human found to stimulate pola at other ratios of poly(dA)oligo(dT) DNA polymerases thought to be involved in DNA replica- or when singly primed 4X174 DNA was used as a template. was tion. Stoichiometric quantities ofhuman SSB are required for The average length of the pola reaction products in- activity, and, perhaps most importantly, it is essential for creased 5- to 10-fold by the addition of human SSB (data not SV40 DNA replication in vitro. Human SSB appears to shown). Although this is consistent with an increase in pola satisfy many criteria expected of a bona fide SSB as exem- processivity, further experiments are required to establish plified by the prototypical T4g32 and E. coli SSB. As pro- the mechanism of stimulation by human SSB. The effect of posed by Chase and Williams (37), once a protein has met SSB on DNA primase has also been examined using poly(dT) these criteria, it should be designated an SSB and the source as the template. Human SSB had little effect on DNA primase of the protein should be indicated. Thus, we believe that activity whereas E. coli SSB inhibited primase activity (data human SSB is more appropriate and informative than other not shown). designations. Human SSB was shown to stimulate pol8, but this effect Human SSB has been shown to support the TAg-catalyzed was not specific since the viral and prokaryotic SSBs also unwinding of SV40 origin-containing DNA (22-24, 26). The worked. This suggests that the mechanism by which human SSB requirement for this reaction appears to be relatively- SSB stimulates pola and pol8 may be different. In fact, the nonspecific since other SSBs (E. coli, Ad DBP, and ICP8) can length of the products obtained with pol8 was not increased substitute for human SSB. The function of the SSB in this by human SSR, suggesting that the association with primer reaction is presumably to stabilize the single-stranded DNA ends rather than processivity may be affected (data not as it is generated by the TAg helicase activity. It is somewhat shown). Although the SSBs tested stimulated pol8 in the surprising that T4g32 and UP1 were not able to function in presence of PCNA and the elongation inhibitor/activator I this assay since both have been shown to lower the melting fraction, the SSBs inhibited DNA synthesis in the absence of temperature of poly(dA-dT) (40, 43). Conversely, Ad DBP the elongation inhibitor/activator I fraction. These observa- supported SV40 DNA unwinding, but it does not markedly tions are analogous to the properties of DNA polymerase III !ower the melting temperature of poly(dA-dT) (44). (pol III) ofE. coli. Pol III holoenzyme is stimulated by E. coli Others have reported (31, 45) that an additional factor(s) is SSB whereas pol III core is inhibited (52, 53). Pol III core has required for efficient DNA unwinding by TAg, SSB, and topo low processivity but is converted to the highly processive pol I. We observe high levels ofunwinding, yet we do not believe III holoenzyme by the addition of accessory subunits. The that the TAg, SSB, or topo I used in these experiments is processivity is dependent on ATP (36, 54, 55). Likewise, contaminated by another factor(s). HeLa topo I can be optimal polb activity requires PCNA, the elongation inhibi- replaced by HeLa topo II or recombinant vaccinia virus topo tor/activator I fraction, human SSB, and ATP. I isolated from E. coli (data not shown). Human SSB, Ad It remains to be proven whether the stimulation ofpola and DBP, ICP8, and E. coli SSB worked efficiently in the DNA pol8 by human SSB is crucial to its role in SV40 DNA unwinding reaction. Immunoaffinity-purified TAg from replication. However, experiments using monoclonal anti- SV40-infected COS cells or from recombinant baculovirus- bodies against the human SSB support this hypothesis (un- infected insect cells were equally active in DNA unwinding, published data). Three anti-SSB monoclonal antibodies that as well as all other reactions catalyzed by TAg. The TAg inhibited SV40 DNA replication also inhibited the pola remained active when subjected to additional purification stimulation but had no effect on DNA unwinding and pol8 steps. However, we have found that TAg isolated from stimulation. It is interesting to note that two of these anti- recombinant adenovirus-infected cells (as used by others) bodies recognized epitopes on the 70-kDa subunit, whereas was inactive in both DNA unwinding and DNA replication the third antibody reacted with the 34-kDa subunit. A fourth when purified proteins were used. This inactive TAg could be antibody, which reacted with the 70-kDa SSB subunit, in- activated by the addition of crude fractions. Virshup and hibited pol8 stimulation and the DNA unwinding reaction but Kelly (31) reported that TAg, prepared from cells infected had no effect on the stimulation of pola by human SSB. with recombinant adenovirus, required a fraction containing protein phosphatase 2A for replication and unwinding activ- We thank Nilda Belgado, Barbara Phillips, and Claudette Turck for ity. Perhaps adenovirus infection leads to TAg phosphoryl- their assistance. We appreciate the generous gifts of vaccinia virus ation and inactivation, which would explain the requirement topo, E. coli SSB, ICP8, and UP1 from Drs. Stewart Shuman, for an additional fraction observed by other laboratories. Kenneth Marians, Michael O'Donnell, and Bruce Alberts, respec- Downloaded by guest on September 28, 2021 Biochemistry: Kenny et al. Proc. Natl. Acad. Sci. USA 86 (1989) 9761 tively. This work was supported by Grant GM34559 from the 27. Bullock, P., Seo, Y. S. & Hurwitz, J. (1989) Proc. Natl. Acad. National Institutes of Health. Sci. USA 86, 3944-3948. 28. Lee, S.-H., Ishimi, Y., Kenny, M. K., Bullock, P., Dean, F. B. 1. Stillman, B. (1989) Annu. Rev. Cell Biol. 5, 197-245. & Hurwitz, J. (1988) Proc. Natl. Acad. Sci. USA 85,9469-9473. 2. Challberg, M. D. & Kelly, T. J. (1989) Annu. Rev. Biochem. 58, 29. Prelich, G. & Stillman, B. (1988) Cell 53, 117-126. 30. Downey, K. M., Tan, C.-K., Andrews, D. M., Li, X. & So, 671-717. A. G. (1988) in Cancer Cells 6, eds. 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