Human DNA polymerase (hPol) functions in the error-free replication of UV-damaged DNA, and mutations in hPol cause cancer-prone syndrome, the variant form of xeroderma pigmentosum. However, in spite of its key role in promoting replication through a variety of distorting DNA lesions, the manner by which hPol is targeted to the replication machinery stalled at a lesion site remains unknown. Here, we provide evidence for the physical interaction of hPol with proliferating cell nuclear antigen (PCNA) and show that mutations in the PCNA binding motif of hPol inactivate this interaction. PCNA, together with replication factor C and replication protein A, stimulates the DNA synthetic activity of hPol, and steady-state kinetic studies indicate that this stimulation accrues from an increase in the efficiency of nucleotide insertion resulting from a reduction in the apparent K m for the incoming nucleotide.DNA polymerase (Pol) is unique among eukaryotic DNA polymerases in its proficient ability to replicate through distorting DNA lesions. Both in yeast and in humans, Pol functions in the error-free replication of UV-damaged DNA (19,26,34,39), and mutations in human Pol (hPol) result in cancer-prone syndrome, the variant form of xeroderma pigmentosum (XP-V) (17, 25). Interestingly, both yeast Pol and hPol replicate through a cis-syn thymine-thymine (TT) dimer with the same efficiency and accuracy as they replicate through undamaged T's (18, 21, 37). Also, genetic studies with yeast have indicated a role for Pol in the error-free bypass of cyclobutane pyrimidine dimers that are formed at 5Ј-TC-3Ј and 5Ј-CC-3Ј sites (40). Pol also promotes replication through a (6-4) TT photoproduct, a highly distorting DNA lesion, by preferentially inserting a G residue opposite the 3Ј T of the photoproduct. Subsequently, Pol efficiently promotes extension from the G residue by inserting the correct nucleotide, A, opposite the 5Ј T of the lesion (16). Although the insertion of a G opposite the 3Ј T of the (6-4) TT photoproduct would cause 3Ј T3C substitutions, it was previously suggested that a similar insertion of G by Pol opposite the 3Ј C of the 5Ј-TC-3Ј and 5Ј-CC-3Ј (6-4) photoproducts, followed by extension by Pol by the insertion of the correct nucleotide opposite the 5Ј residue of this lesion, would lead to error-free bypass of the DNA lesion (16). Since (6-4) photoproducts are formed much more frequently at TC and CC sites than at TT sites (4, 6), Pol would largely contribute to the error-free bypass of (6-4) lesions as well. Yeast Pol and hPol also efficiently replicate through other DNA lesions, such as 8-oxoguanine (15) and O 6 -methylguanine (13).The ability of Pol to replicate through distorting DNA lesions has suggested that the active site of Pol is tolerant of geometric distortions introduced into DNA by these lesions. As a consequence, Pol is a low-fidelity enzyme, and on undamaged DNA, the yeast and human enzymes misincorporate nucleotides with a frequency of 10 Ϫ2 to 10 Ϫ3 (21, 38). In sharp contrast, replicative DNA pol...
The replication of bovine papilloma virus (BPV) DNA in vivo requires two viral-encoded proteins, El and E2, while all other proteins are derived from the host. We described previously the isolation of the El protein and showed that it contains multiple functions required for BPV DNA replication. The BPV transcription factor E2 was shown by others to stimulate BPV DNA replication in vitro. Here, we present results that account for the role of the E2 protein. The El protein bound selectively to the BPV minimal origin of replication. This process required MgCl2 and ATP for maxal effi'ciency. The El protein also catalyzed a BPV origindependent DNA unwinding reaction. In this report, we show that at low levels of El protein, origin binding could be stimulated up to 40-fold by the E2 protein, provided that the DNA contained an E2 binding site. Consistent with this result, the E2 protein stimulated the origin-specific unwinding reaction catalyzed by El, but it had no effect on the nonspecific El-catalyzed helicase activity. In the absence of an E2 binding site, both origin-dependent binding and unwinding reactions with the El protein were unaffected by the E2 protein. These results suggest that E2 participates in the initiation of BPV DNA replication by enhancing El binding to the BPV or4ign via DNA-protein and protein-protein interactions.Bovine papilloma virus (BPV) provides an attractive model system to study the regulation of eukaryotic DNA replication. The viral DNA is maintained in BPV transformed cells as a nuclear plasmid with a constant copy number (1). Recent studies indicate that in vivo BPV DNA replication requires two BPV viral-encoded proteins-the 68-kDa El protein and the 48-kDa E2 protein (2). In addition, the minimal origin sequence that supports BPV DNA replication has been identified (nt 7911-22 of the BPV type 1 genome) (3,4). This sequence contains a binding site for the El protein (El BS) and part of a sequence that acts as a binding site for the E2 protein (E2 BS).The replication of BPV oril DNA in vitro, using extracts of a mouse mammary tumor cell line (FM3A), was recently established in Botchan's laboratory (4). They demonstrated that DNA synthesis required the BPV minimal origin and the El protein, whereas all other required proteins were supplied by uninfected mouse cell extracts. At low concentrations of El protein, the replication reaction was stimulated markedly by the E2 protein (4).We have described the isolation of the El protein and showed that it supported BPV ori+ DNA replication in vitro (5). We also demonstrated that this protein possessed a number of different activities required for BPV DNA replication. These include (i) a DNA helicase activity, for which the protein translocates in the 3' to 5' direction, (ii) a BPV oril DNA binding activity that is stimulated by ATP and MgCl2, and (iii) the capacity to unwind covalently closed circular ori+ DNA leading to highly unwound DNA products (5). Thus, the role of El in the BPV system is analogous to that of large tumor antigen...
Human DNA polymerase (hPol) promotes translesion synthesis by inserting nucleotides opposite highly distorting or noninstructional DNA lesions. Here, we provide evidence for the physical interaction of hPol with proliferating cell nuclear antigen (PCNA), and show that PCNA, together with replication factor C (RFC) and replication protein A (RPA), stimulates the DNA synthetic activity of hPol. In the presence of these protein factors, on undamaged DNA, the efficiency (Vmax͞Km) of correct nucleotide incorporation by hPol is increased Ϸ80 -150-fold, and this increase in efficiency results from a reduction in the apparent Km for the nucleotide. PCNA, RFC, and RPA also stimulate nucleotide incorporation opposite the 3-T of the (6 -4) thymine-thymine (T-T) photoproduct and opposite an abasic site. The interaction of hPol with PCNA implies that the targeting of this polymerase to the replication machinery stalled at a lesion site is achieved via this association.
Humans have three DNA polymerases, Pol, Pol, and Pol, which are able to promote replication through DNA lesions. However, the mechanism by which these DNA polymerases are targeted to the replication machinery stalled at a lesion site has remained unknown. Here, we provide evidence for the physical interaction of human Pol (hPol) with proliferating cell nuclear antigen (PCNA) and show that PCNA, replication factor C (RFC), and replication protein A (RPA) act cooperatively to stimulate the DNA synthesis activity of hPol. The processivity of hPol, however, is not significantly increased in the presence of these protein factors. The efficiency (V max /K m ) of correct nucleotide incorporation by hPol is enhanced ϳ50-to 200-fold in the presence of PCNA, RFC, and RPA, and this increase in efficiency is achieved by a reduction in the apparent K m for the nucleotide. Although in the presence of these protein factors, the efficiency of the insertion of an A nucleotide opposite an abasic site is increased ϳ40-fold, this reaction still remains quite inefficient; thus, it is unlikely that hPol would bypass an abasic site by inserting a nucleotide opposite the site.In both prokaryotes and eukaryotes, DNA polymerases belonging to the UmuC/Rad30/DinB family promote replication through DNA lesions (6,16). In eukaryotes, DNA polymerase (Pol) functions in the error-free replication of UV-damaged DNA, and both yeast Pol and human Pol (hPol) replicate through a cis-syn thymine-thymine (T-T) dimer with the same efficiency and accuracy with which they replicate through undamaged T's (13,17,31). Genetic studies of yeast have also implicated Pol in the error-free bypass of cyclobutane dimers formed at 5Ј-TC-3Ј and 5Ј-CC-3Ј sites (32), and Pol efficiently replicates through many other DNA lesions as well (9,11). Mutations in Pol in humans cause a cancer-prone syndrome, the variant form of xeroderma pigmentosum (12,22).In addition to Pol, humans contain two other related polymerases, Pol and Pol (14,15,25,30). Unlike Pol, Pol does not bypass a cis-syn T-T dimer and it does not even insert a nucleotide opposite the 3Ј T of the dimer (15). A role of Pol in lesion bypass, however, is indicated by its ability to incorporate nucleotides opposite the 3Ј T of the (6-4) T-T photoproduct and opposite abasic sites (15). Subsequently, Pol extends from the nucleotide inserted by Pol, thus completing the bypass process (15).In contrast to Pol and Pol, which exist only in eukaryotes, the DinB protein and its homologs are found in both prokaryotes and eukaryotes (6,16). In Escherichia coli, dinB-encoded Pol IV shows little ability to bypass a cis-syn T-T dimer, a (6-4) T-T photoproduct, or an abasic site (29). However, genetic studies have implicated Pol IV in the mutagenic replication of undamaged DNA, as deletion of the dinB gene in E.coli reduces the rates of frameshift and base substitution mutations. This effect of dinB in generating spontaneous mutations becomes more evident when the replicative polymerase has been partially disabled and the mism...
Human replication factor C (RFC, also called activator 1) is a five-subunit protein complex (p140, p40, p38, p37, and p36) required for proliferating cell nuclear antigen (PCNA)-dependent processive DNA synthesis catalyzed by DNA polymerase ␦ or . Here we report the reconstitution of the RFC complex from its five subunits simultaneously overexpressed in baculovirus-infected insect cells. The purified baculovirus-produced RFC appears to contain equimolar levels of each subunit and was shown to be functionally identical to its native counterpart in (i) supporting DNA polymerase ␦-catalyzed PCNA-dependent DNA chain elongation; (ii) catalyzing DNA-dependent ATP hydrolysis that was stimulated by PCNA and human single-stranded DNA binding protein; (iii) binding preferentially to DNA primer ends; and (iv) catalytically loading PCNA onto singly nicked circular DNA and catalytically removing PCNA from these DNA molecules.Replication factor C (RFC; also known as activator 1) functions as an accessory factor for proliferating cell nuclear antigen (PCNA)-dependent DNA synthesis catalyzed by DNA polymerase ␦ or (pol ␦ or ) (1-7). RFC contains multiple activities including its ability to preferentially bind DNA primer ends and catalyze DNA-dependent ATP hydrolysis.Following its association with DNA at a primer end, RFC recruits PCNA (the clamp) and loads it onto DNA in the presence of ATP (clamp loading) (8,9). This complex then tethers pol ␦ to the DNA primer junction in a reaction that requires ATP hydrolysis and results in highly processive DNA chain elongation (1-7, 10). This RFC-dependent PCNA loading mechanism is conserved among three species examined (human, Escherichia coli, and T4 bacteriophage). In E. coli and T4, the functional homologs of RFC are the ␥ complex and T4 gene products (gp) 44͞62, while the counterparts of PCNA are the  subunit of the pol III holoenzyme and gp45, respectively.In E. coli, the  clamp remains on DNA after completion of DNA synthesis and dissociation of the DNA polymerase. It is likely that a similar mechanism occurs in eukaryotes. In E. coli and HeLa cells, the estimated number of Okazaki fragments formed during one round of replication is Ͼ10 and 100 ϫ the amounts of  and PCNA, respectively. As a result, the clamps must be recycled to ensure continuous DNA synthesis. The clamp loaders, the ␥ complex in E. coli and RFC in eukaryotes, also carry out the unloading of clamps to recycle these proteins. However, in T4 bacteriophage, clamp unloading may not be an active process but results from the spontaneous dissociation of gp45 as a result of its intrinsic instability on DNA (for summary, see ref. 8).RFC is highly conserved from yeast (sc) to humans (h) in its subunit structure. It contains five subunits ranging between 36-140 kDa as revealed by SDS͞PAGE. Genes encoding each of these subunits have been cloned from both mammals (12-17) and Saccharomyces cerevisiae, and each subunit has been shown to be essential following deletion analysis in yeast (18)(19)(20)(21)(22)(23). The pre...
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