The Many Roles of PCNA in Eukaryotic DNA Replication.

Proliferating cell nuclear antigen (PCNA) plays critical roles in many aspects of DNA replication and replication-associated processes, including translesion synthesis, error-free damage bypass, break-induced replication, mismatch repair, and chromatin assembly. Since its discovery, our view of PCNA has evolved from a replication accessory factor to the hub protein in a large protein-protein interaction network that organizes and orchestrates many of the key events at the replication fork. We begin this review article with an overview of the structure and function of PCNA. We discuss the ways its many interacting partners bind and how these interactions are regulated by posttranslational modifications such as ubiquitylation and sumoylation. We then explore the many roles of PCNA in normal DNA replication and in replication-coupled DNA damage tolerance and repair processes. We conclude by considering how PCNA can interact physically with so many binding partners to carry out its numerous roles. We propose that there is a large, dynamic network of linked PCNA molecules at and around the replication fork. This network would serve to increase the local concentration of all the proteins necessary for DNA replication and replication-associated processes and to regulate their various activities.

Fidelity and damage bypass ability of Schizosaccharomyces pombe Eso1 protein, comprised of DNA polymerase eta and sister chromatid cohesion protein Ctf7.

DNA polymerase eta (Poleta) functions in error-free bypass of ultraviolet light-induced DNA lesions, and mutational inactivation of Poleta in humans causes the cancer prone syndrome, the variant form of xeroderma pigmentosum (XPV). Both Saccharomyces cerevisiae and human Poleta efficiently insert two adenines opposite the two thymines of a cyclobutane pyrimidine dimer. Interestingly, in the fission yeast Schizosaccharomyces pombe, the eso1(+) encoded protein is comprised of two domains, wherein the NH(2) terminus is highly homologous to Poleta, and the COOH terminus is highly homologous to the S. cerevisiae Ctf7 protein which is essential for the establishment of sister chromatid cohesion during S phase. Here we characterize the DNA polymerase activity of S. pombe GST-Eso1 fusion protein and a truncated version containing only the Poleta domain. Both proteins exhibit a similar DNA polymerase activity with a low processivity, and steady-state kinetic analyses show that on undamaged DNA, both proteins misincorporate nucleotides with frequencies of approximately 10(-2) to 10(-3). We also examine the two proteins for their ability to replicate a cyclobutane pyrimidine dimer-containing DNA template and find that both proteins replicate through the lesion equally well. Thus, fusion with Ctf7 has no significant effect on the DNA replication or damage bypass properties of Poleta. The possible role of Ctf7 fusion with Poleta in the replication of Cohesin-bound DNA sequences is discussed.

Accuracy of lesion bypass by yeast and human DNA polymerase eta.

DNA polymerase eta (Pol eta) functions in the error-free bypass of UV-induced DNA lesions, and a defect in Pol eta in humans causes the cancer-prone syndrome, the variant form of xeroderma pigmentosum. Both yeast and human Pol eta replicate through a cis-syn thymine-thymine dimer (TT dimer) by inserting two As opposite the two Ts of the dimer. Pol eta, however, is a low-fidelity enzyme, and it misinserts nucleotides with a frequency of approximately 10(-2) to 10(-3) opposite the two Ts of the TT dimer as well as opposite the undamaged template bases. This low fidelity of nucleotide insertion seems to conflict with the role of Pol eta in the error-free bypass of UV lesions. To resolve this issue, we have examined the ability of human and yeast Pol eta to extend from paired and mispaired primer termini opposite a TT dimer by using steady-state kinetic assays. We find that Pol eta extends from mispaired primer termini on damaged and undamaged DNAs with a frequency of approximately 10(-2) to 10(-3) relative to paired primer termini. Thus, after the incorporation of an incorrect nucleotide, Pol eta would dissociate from the DNA rather than extend from the mispair. The resulting primer-terminal mispair then could be subject to proofreading by a 3'-->5' exonuclease. Replication through a TT dimer by Pol eta then would be more accurate than that predicted from the fidelity of nucleotide incorporation alone.

Acidic residues critical for the activity and biological function of yeast DNA polymerase eta.

Rad30 is a member of the newly discovered UmuC/DinB/Rad30 family of DNA polymerases. The N-terminal regions of these proteins are highly homologous, and they contain five conserved motifs, I to V, while their C-terminal regions are quite divergent. We examined the contributions of the C-terminal and N-terminal regions of Rad30 to its activity and biological function. Although deletion of the last 54 amino acids has no effect on DNA polymerase or thymine-thymine (T-T) dimer bypass activity, this C-terminal deletion-containing protein is unable to perform its biological function in vivo. The presence of a bipartite nuclear targeting sequence within this region suggests that at least one function of this portion of Rad30 is nuclear targeting. To identify the active-site residues of Rad30 important for catalysis, we generated mutations of nine acidic residues that are invariant or highly conserved among Rad30 proteins from different eukaryotic species. Mutations of the Asp30 and Glu39 residues present in motif I and of the Asp155 residue present in motif III to alanine completely inactivated the DNA polymerase and T-T dimer bypass activities, and these mutations did not complement the UV sensitivity of the rad30Delta mutation. Mutation of Glu156 in motif III to alanine confers a large reduction in the efficiency of nucleotide incorporation, whereas the remaining five Rad30 mutant proteins retain wild-type levels of DNA polymerase and T-T dimer bypass activities. From these observations, we suggest a role for the Asp30, Glu39, and Asp155 residues in the binding of two metal ions required for the reaction of the incoming deoxynucleoside 5'-triphosphate with the 3'-hydroxyl in the primer terminus, while Glu156 may participate in nucleotide binding.

Inefficient bypass of an abasic site by DNA polymerase eta.

DNA polymerase eta (Pol eta) bypasses a cis-syn thymine-thymine dimer efficiently and accurately, and inactivation of Pol eta in humans results in the cancer-prone syndrome, the variant form of xeroderma pigmentosum. Also, Pol eta bypasses the 8-oxoguanine lesion efficiently by predominantly inserting a C opposite this lesion, and it bypasses the O(6)-methylguanine lesion by inserting a C or a T. To further assess the range of DNA lesions tolerated by Pol eta, here we examine the bypass of an abasic site, a prototypical noninstructional lesion. Steady-state kinetic analyses show that both yeast and human Pol eta are very inefficient in both inserting a nucleotide opposite an abasic site and in extending from the nucleotide inserted. Hence, Pol eta bypasses this lesion extremely poorly. These results suggest that Pol eta requires the presence of template bases opposite both the incoming nucleotide and the primer terminus to catalyze efficient nucleotide incorporation.

Mismatch extension ability of yeast and human DNA polymerase eta.

DNA polymerase eta (Poleta) functions in error-free replication of UV-damaged DNA, and in vitro it efficiently bypasses a cis-syn T-T dimer by incorporating two adenines opposite the lesion. Steady state kinetic studies have shown that both yeast and human Poleta are low-fidelity enzymes, and they misincorporate nucleotides with a frequency of 10(-2)-10(-3) on both undamaged and T-T dimer-containing DNA templates. To better understand the role of Poleta in error-free translesion DNA synthesis, here we examine the ability of Poleta to extend from base mismatches. We find that both yeast and human Poleta extend from mismatched base pairs with a frequency of approximately 10(-3) relative to matched base pairs. In the absence of efficient extension of mismatched primer termini, the ensuing dissociation of Poleta from DNA may favor the excision of mismatched nucleotides by a proofreading exonuclease. Thus, we expect DNA synthesis by Poleta to be more accurate than that predicted from the fidelity of nucleotide incorporation alone.

Translesion DNA synthesis by yeast DNA polymerase eta on templates containing N2-guanine adducts of 1,3-butadiene metabolites.

Yeast DNA polymerase eta can replicate through cis-syn cyclobutane pyrimidine dimers and 8-oxoguanine lesions with the same efficiency and accuracy as replication of an undamaged template. Previously, it has been shown that Escherichia coli DNA polymerases I, II, and III are incapable of bypassing DNA substrates containing N(2)-guanine adducts of stereoisomeric 1,3-butadiene metabolites. Here we showed that yeast polymerase eta replicates DNA containing the monoadducts (S)-butadiene monoepoxide and (S,S)-butadiene diolepoxide N(2)-guanines albeit at an approximately 200-300-fold lower efficiency relative to the control guanine. Interestingly, nucleotide incorporation opposite the (R)-butadiene monoepoxide and the (R,R)-butadiene diolepoxide N(2)-guanines was approximately 10-fold less efficient than incorporation opposite their S stereoisomers. Polymerase eta preferentially incorporates the correct nucleotide opposite and downstream of all four adducts, except that it shows high misincorporation frequencies for elongation of C paired with (R)-butadiene monoepoxide N(2)-guanine. Additionally, polymerase eta does not bypass the (R,R)- and (S,S)-butadiene diolepoxide N(2)-guanine-N(2)-guanine intra- strand cross-links, and replication is completely blocked just prior to the lesion. Collectively, these data suggest that polymerase eta can tolerate the geometric distortions in DNA conferred by the N(2)-guanine butadiene monoadducts but not the intrastrand cross-links.

Yeast DNA polymerase eta utilizes an induced-fit mechanism of nucleotide incorporation.

DNA polymerase eta (Poleta) is unique among eukaryotic DNA polymerases in its proficient ability to replicate through distorting DNA lesions, and Poleta synthesizes DNA with a low fidelity. Here, we use pre-steady-state kinetics to investigate the mechanism of nucleotide incorporation by Poleta and show that it utilizes an induced-fit mechanism to selectively incorporate the correct nucleotide. Poleta discriminates poorly between the correct and incorrect nucleotide at both the initial nucleotide binding step and at the subsequent induced-fit conformational change step, which precedes the chemical step of phosphodiester bond formation. This property enables Poleta to bypass lesions with distorted DNA geometries, and it bestows upon the enzyme a low fidelity.

Eukaryotic polymerases iota and zeta act sequentially to bypass DNA lesions.

DNA lesions can often block DNA replication, so cells possess specialized low-fidelity, and often error-prone, DNA polymerases that can bypass such lesions and promote replication of damaged DNA. The Saccharomyces cerevisiae RAD30 and human hRAD30A encode Pol eta, which bypasses a cis-syn thymine-thymine dimer efficiently and accurately. Here we show that a related human gene, hRAD30B, encodes the DNA polymerase Pol iota, which misincorporates deoxynucleotides at a high rate. To bypass damage, Pol iota specifically incorporates deoxynucleotides opposite highly distorting or non-instructional DNA lesions. This action is combined with that of DNA polymerase Pol zeta, which is essential for damage-induced mutagenesis, to complete the lesion bypass. Pol zeta is very inefficient in inserting deoxynucleotides opposite DNA lesions, but readily extends from such deoxynucleotides once they have been inserted. Thus, in a new model for mutagenic bypass of DNA lesions in eukaryotes, the two DNA polymerases act sequentially: Pol iota incorporates deoxynucleotides opposite DNA lesions, and Pol zeta functions as a mispair extender.

Fidelity of human DNA polymerase eta.

Xeroderma pigmentosum (XP) patients are highly sensitive to sunlight, and they suffer from a high incidence of skin cancers. The variant form of XP results from mutations in the hRAD30A gene, which encodes the DNA polymerase in humans, hPol(eta). Of the eukaryotic DNA polymerases, only human Pol(eta) and its yeast counterpart have the ability to replicate DNA containing a cis-syn thymine-thymine (T-T) dimer. Here we measure the fidelity of hPol(eta) on all four nondamaged template bases and at each thymine residue of a cis-syn T-T dimer. Opposite all four nondamaged template bases, hPol(eta) misincorporates nucleotides with a frequency of approximately 10(-2)-10(-3), and importantly, hPol(eta) synthesizes DNA opposite the T-T dimer with the same accuracy and efficiency as opposite the nondamaged DNA. The low fidelity of hPol(eta) may derive from a flexible active site that renders the enzyme more tolerant of geometric distortions in DNA and enables it to synthesize DNA past a T-T dimer.

Accuracy of thymine-thymine dimer bypass by Saccharomyces cerevisiae DNA polymerase eta.

The Saccharomyces cerevisiae RAD30 gene functions in error-free replication of UV-damaged DNA. RAD30 encodes a DNA polymerase, Pol eta, which inserts two adenines opposite the two thymines of a cis-syn thymine-thymine (T-T) dimer. Here we use steady-state kinetics to determine the accuracy of DNA synthesis opposite the T-T dimer. Surprisingly, the accuracy of DNA synthesis opposite the damaged DNA is nearly indistinguishable from that opposite nondamaged DNA, with frequencies of misincorporation of about 10(-2) to 10(-3). These studies support the hypothesis that unlike most DNA polymerases, Pol eta is able to tolerate distortions in DNA resulting from damage, which then enables the polymerase to utilize the intrinsic base pairing ability of the T-T dimer.

Fidelity and processivity of Saccharomyces cerevisiae DNA polymerase eta.

The yeast RAD30 gene functions in error-free replication of UV-damaged DNA, and RAD30 encodes a DNA polymerase, pol eta, that has the ability to efficiently and correctly replicate past a cis-syn-thymine-thymine dimer in template DNA. To better understand the role of pol eta in damage bypass, we examined its fidelity and processivity on nondamaged DNA templates. Steady-state kinetic analyses of deoxynucleotide incorporation indicate that pol eta has a low fidelity, misincorporating deoxynucleotides with a frequency of about 10(-2) to 10(-3). Also pol eta has a low processivity, incorporating only a few nucleotides before dissociating. We suggest that pol eta's low fidelity reflects a flexibility in its active site rendering it more tolerant of DNA damage, while its low processivity limits its activity to reduce errors.

Increased DNA unwinding efficiency of bacteriophage T7 DNA helicase mutant protein 4A'/E348K.

Bacteriophage T7 4A' protein is a DNA helicase that unwinds DNA in a reaction coupled to dTTP hydrolysis. To understand better its mechanism of DNA unwinding, we characterized a set of 4A' mutant proteins (Washington, M. T., Rosenberg, A. H., Griffin, K., Studier, F. W., and Patel, S. S. (1996) J. Biol. Chem. 271, 26825-26834). We showed here, using single turnover DNA unwinding assays, that the 4A'/E348K mutant protein had the unusual property of unwinding DNA (with a 5-6-fold slower rate) despite a significant defect in its dTTPase activity (a 25-30-fold slower rate). Comparing the DNA unwinding rates to the dTTPase rates, we estimated the DNA unwinding efficiencies of both wild-type (about 1 base pair unwound per dTTP hydrolysis) and mutant (4 to 6 base pairs unwound per dTTP hydrolysis). Thus the mutant had a 4-6-fold improvement in its DNA unwinding efficiency over that of the wild-type. We believe that this mutant undergoes less slippage (uncoupled dTTP hydrolysis) than the wild-type. We speculate that nature has selected for a high rate of DNA unwinding rather than a high efficiency of DNA unwinding. Thus even though the mutant is more efficient at DNA unwinding, the wild-type probably was selected because it unwinds DNA faster.

The dTTPase mechanism of T7 DNA helicase resembles the binding change mechanism of the F1-ATPase.

Bacteriophage T7 DNA helicase is a ring-shaped hexamer that catalyzes duplex DNA unwinding using dTTP hydrolysis as an energy source. Of the six potential nucleotide binding sites on the hexamer, we have found that three are noncatalytic sites and three are catalytic sites. The noncatalytic sites bind nucleotides with a high affinity, but dTTPs bound to these sites do not dissociate or hydrolyze through many dTTPase turnovers at the catalytic sites. The catalytic sites show strong cooperativity which leads to sequential binding and hydrolysis of dTTP. The elucidated dTTPase mechanism of the catalytic sites of T7 helicase is remarkably similar to the binding change mechanism of the ATP synthase. Based on the similarity, a general mechanism for hexameric helicases is proposed. In this mechanism, an F1-ATPase-like rotational movement around the single-stranded DNA, which is bound through the central hole of the hexamer, is proposed to lead to unidirectional translocation along single-stranded DNA and duplex DNA unwinding.

Selection, identification, and genetic analysis of random mutants in the cloned primase/helicase gene of bacteriophage T7.

T7 gene 4 specifies two overlapping proteins 4A, a 566-amino acid primase/helicase, and 4B, a 503-amino acid helicase whose initiation codon is the 64th codon of the 4A protein. The 4A' gene, which has a leucine codon replacing the 4B initiation codon, specifies a single 566-amino acid protein that can provide the primase and helicase functions required for normal T7 growth. We selected N-methyl-N'-nitro-N-nitrosoguanidine mutants in the cloned 4A' gene that no longer support the growth of a phage that completely lacks gene 4. Genetic mapping of the 76 mutations found them to be distributed throughout the protein, including both the N-terminal and C-terminal halves of the molecule thought to represent primase and helicase domains, respectively. Complementation tests with partially and completely defective phage showed that all but five of the mutants lacked helicase function but retained primase function. The other five, which lacked both functions, all made short proteins, including one missing only 60 amino acids. No mutations lacked only primase function, and none mapped within the first 105 amino acids, which includes the 63-amino acid region unique to 4A that contains elements required to recognize primase sites. Forty-six mutations were sequenced and included 27 missense mutations affecting 25 amino acids. Many mutations in the N-terminal half of the protein affected its solubility in cell extracts. Mutations in the C-terminal half clustered in or near five helicase consensus sequences. Biochemical analysis of nine of the mutant proteins is described in the accompanying paper (Washington, M. T., Rosenberg, A. H., Griffin, K., Studier, F. W., and Patel, S. S. (1996) J. Biol. Chem. 271, 26825-26834).

Biochemical analysis of mutant T7 primase/helicase proteins defective in DNA binding, nucleotide hydrolysis, and the coupling of hydrolysis with DNA unwinding.

We characterized nine helicase-deficient mutants of bacteriophage T7 helicase-primase protein (4A') prepared by random mutagenesis as reported in the accompanying paper (Rosenberg, A. H., Griffin, K., Washington, M. T., Patel, S. S., and Studier, F. W. (1996) J. Biol. Chem. 271, 26819-26824). Mutants were selected from each of the helicase-conserved motifs for detailed analysis to understand better their function. In agreement with the in vivo results, the mutants were defective in helicase activity but were active in primase function. dTTP hydrolysis, DNA binding, and hexamer formation were examined. Three classes of defective mutants were observed. Group A mutants (E348K, D424N, and S496F), defective in dTTP hydrolysis, lie in motifs 1a, 2, and 4 and are possibly involved in NTP binding/hydrolysis. Group B mutants (R487C and G488D), defective in DNA binding, lie in motif 4 and are responsible directly or indirectly for DNA binding. Group C mutants (G116D, A257T, S345F, and G451E) were not defective in any of the activities except the helicase function. These mutants, scattered throughout the protein, appear defective in coupling dTTPase activity to helicase function. Secondary structural predictions of 4A' and DnaB helicases resemble the known structures of RecA and F1-ATPase enzymes. Alignment shows a striking correlation in the positions of the amino acids that interact with NTP and DNA.