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DNA polymerase η (Polη) is a translesion synthesis polymerase that can bypass different DNA lesions with varying efficiency and fidelity. Its most well-known function is the error-free bypass of ultraviolet light-induced cyclobutane pyrimidine dimers. The lack of this unique ability in humans leads to the development of a cancer-predisposing disease, the variant form of xeroderma pigmentosum. Human Polη can insert rNTPs during DNA synthesis, though with much lower efficiency than dNTPs, and it can even extend an RNA chain with ribonucleotides. We have previously shown that Mn2+ is a specific activator of the RNA synthetic activity of yeast Polη that increases the efficiency of the reaction by several thousand-fold over Mg2+. In this study, our goal was to investigate the metal cofactor dependence of RNA synthesis by human Polη. We found that out of the investigated metal cations, only Mn2+ supported robust RNA synthesis. Steady state kinetic analysis showed that Mn2+ activated the reaction a thousand-fold compared to Mg2+, even during DNA damage bypass opposite 8-oxoG and TT dimer. Our results revealed a two order of magnitude higher affinity of human Polη towards ribonucleotides in the presence of Mn2+ compared to Mg2+. It is noteworthy that activation occurred without lowering the base selectivity of the enzyme on undamaged templates, whereas the fidelity decreased across a TT dimer. In summary, our data strongly suggest that, like with its yeast homolog, Mn2+ is the proper metal cofactor of hPolη during RNA chain extension, and selective metal cofactor utilization contributes to switching between its DNA and RNA synthetic activities.

PCNA is a central orchestrator of cellular processes linked to DNA metabolism. It is a binding platform for a plethora of proteins and coordinates and regulates the activity of several pathways. The outer side of PCNA comprises most of the known interacting and regulatory surfaces, whereas the residues at the inner side constitute the sliding surface facing the DNA double helix. Here, by investigating the L154A mutation found at the inner side, we show that the inner surface mediates protein interactions essential for genome stability. It forms part of the binding site of Rad18, a key regulator of DNA damage tolerance, and is required for PCNA sumoylation which prevents unscheduled recombination during replication. In addition, the L154 residue is necessary for stable complex formation between PCNA and the replicative DNA polymerase δ. Hence, its absence increases the mutation burden of yeast cells due to faulty replication. In summary, the essential role of the L154 of PCNA in guarding and maintaining stable replication and promoting DNA damage tolerance reveals a new connection between these processes and assigns a new coordinating function to the central channel of PCNA.

Polymerase eta (Pol&eta, ) is a translesion synthesis DNA polymerase directly linked to cancer development. It can bypass several DNA lesions thereby rescuing DNA damage-stalled replication complexes. We previously presented evidence implicating Saccharomyces cerevisiae Pol&eta, in transcription elongation, and identified its specific RNA extension and translesion RNA synthetic activities. However, RNA synthesis by Pol&eta, proved rather inefficient under conditions optimal for DNA synthesis. Searching for factors that could enhance its RNA synthetic activity, we have identified the divalent cation of manganese. Here, we show that manganese triggers drastic changes in the activity of Pol&eta, Kinetics experiments indicate that manganese increases the efficiency of ribonucleoside incorporation into RNA by ~400&ndash, 2000-fold opposite undamaged DNA, and ~3000 and ~6000-fold opposite TT dimer and 8oxoG, respectively. Importantly, preference for the correct base is maintained with manganese during RNA synthesis. In contrast, activity is strongly impaired, and base discrimination is almost lost during DNA synthesis by Pol&eta, with manganese. Moreover, Pol&eta, shows strong preference for manganese during RNA synthesis even at a 25-fold excess magnesium concentration. Based on this, we suggest that a new regulatory mechanism, selective metal cofactor utilization, modulates the specificity of Pol&eta, helping it to perform distinct activities needed for its separate functions during replication and transcription.

Polymerase eta (Polη) is a low fidelity translesion synthesis DNA polymerase that rescues damage-stalled replication by inserting deoxy-ribonucleotides opposite DNA damage sites resulting in error-free or mutagenic damage bypass. In this study we identify a new specific RNA extension activity of Polη of Saccharomyces cerevisiae. We show that Polη is able to extend RNA primers in the presence of ribonucleotides (rNTPs), and that these reactions are an order of magnitude more efficient than the misinsertion of rNTPs into DNA. Moreover, during RNA extension Polη performs error-free bypass of the 8-oxoguanine and thymine dimer DNA lesions, though with a 103 and 102–fold lower efficiency, respectively, than it synthesizes opposite undamaged nucleotides. Furthermore, in vivo experiments demonstrate that the transcription of several genes is affected by the lack of Polη, and that Polη is enriched over actively transcribed regions. Moreover, inactivation of its polymerase activity causes similar transcription inhibition as the absence of Polη. In summary, these results suggest that the new RNA synthetic activity of Polη can have in vivo relevance.

In the yeast Saccharomyces cerevisiae, the Rad6-Rad18 DNA damage tolerance pathway constitutes a major defense system against replication fork blocking DNA lesions. The Rad6-Rad18 ubiquitin-conjugating/ligase complex governs error-free and error-prone translesion synthesis by specialized DNA polymerases, as well as an error-free Rad5-dependent postreplicative repair pathway. For facilitating replication through DNA lesions, translesion synthesis polymerases copy directly from the damaged template, while the Rad5-dependent damage tolerance pathway obtains information from the newly synthesized strand of the undamaged sister duplex. Although genetic data demonstrate the importance of the Rad5-dependent pathway in tolerating DNA damages, there has been little understanding of its mechanism. Also, the conservation of the yeast Rad5-dependent pathway in higher order eukaryotic cells remained uncertain for a long time. Here we summarize findings published in recent years regarding the role of Rad5 in promoting error-free replication of damaged DNA, and we also discuss results obtained with its human orthologs, HLTF and SHPRH.

Unrepaired DNA lesions can block the progression of the replication fork, leading to genomic instability and cancer in higher-order eukaryotes. In Saccharomyces cerevisiae, replication through DNA lesions can be mediated by translesion synthesis DNA polymerases, leading to error-free or error-prone damage bypass, or by Rad5-mediated template switching to the sister chromatid that is inherently error free. While translesion synthesis pathways are highly conserved from yeast to humans, very little is known of a Rad5-like pathway in human cells. Here we show that a human homologue of Rad5, HLTF, can facilitate fork regression and has a role in replication of damaged DNA. We found that HLTF is able to reverse model replication forks, a process which depends on its double-stranded DNA translocase activity. Furthermore, from analysis of isolated dually labeled chromosomal fibers, we demonstrate that in vivo, HLTF promotes the restart of replication forks blocked at DNA lesions. These findings suggest that HLTF can promote error-free replication of damaged DNA and support a role for HLTF in preventing mutagenesis and carcinogenesis, providing thereby for its potential tumor suppressor role.

Human Ape2 protein has 3′ phosphodiesterase activity for processing 3′-damaged DNA termini, 3′–5′ exonuclease activity that supports removal of mismatched nucleotides from the 3′-end of DNA, and a somewhat weak AP-endonuclease activity. However, very little is known about the role of Ape2 in DNA repair processes. Here, we examine the effect of interaction of Ape2 with proliferating cell nuclear antigen (PCNA) on its enzymatic activities and on targeting Ape2 to oxidative DNA lesions. We show that PCNA strongly stimulates the 3′–5′ exonuclease and 3′ phosphodiesterase activities of Ape2, but has no effect on its AP-endonuclease activity. Moreover, we find that upon hydrogen-peroxide treatment Ape2 redistributes to nuclear foci where it colocalizes with PCNA. In concert with these results, we provide biochemical evidence that Ape2 can reduce the mutagenic consequences of attack by reactive oxygen species not only by repairing 3′-damaged termini but also by removing 3′-end adenine opposite from 8-oxoG. Based on these findings we suggest the involvement of Ape2 in repair of oxidative DNA damage and PCNA-dependent repair synthesis.

Treatment of yeast and human cells with DNA-damaging agents elicits Rad6–Rad18-mediated monoubiquitination of proliferating cell nuclear antigen (PCNA) at its Lys-164 residue [ubiquitin (Ub)-PCNA], and this PCNA modification is indispensable for promoting the access of translesion synthesis (TLS) polymerases (Pols) to PCNA. However, the means by which K164-linked Ub modulates the proficiency of TLS Pols to bind PCNA and take over synthesis from the replicative Pol has remained unclear. One model that has gained considerable credence is that the TLS Pols bind PCNA at 2 sites, to the interdomain connector loop via their PCNA-interacting protein (PIP) domain and to the K164-linked Ub moiety via their Ub-binding domain (UBD). Specifically, this model postulates that the UBD-mediated binding of TLS Pols to the Ub moiety on PCNA is necessary for TLS. To test the validity of this model, we examine the contributions that the PIP and Ub-binding zinc finger (UBZ) domains of human Polη make to its functional interaction with PCNA, its colocalization with PCNA in replication foci, and its role in TLS in vivo. We conclude from these studies that the binding to PCNA via its PIP domain is a prerequisite for Polη's ability to function in TLS in human cells and that the direct binding of the Ub moiety on PCNA via its UBZ domain is not required. We discuss the possible role of the Ub moiety on PCNA in TLS.

Humans have three DNA polymerases, Poleta, Polkappa, and Poliota, 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 Polkappa (hPolkappa) 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 hPolkappa. The processivity of hPolkappa, however, is not significantly increased in the presence of these protein factors. The efficiency (V(max)/K(m)) of correct nucleotide incorporation by hPolkappa is enhanced approximately 50- 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 approximately 40-fold, this reaction still remains quite inefficient; thus, it is unlikely that hPolkappa would bypass an abasic site by inserting a nucleotide opposite the site.

Human DNA polymerase ι (hPolι) promotes translesion synthesis by inserting nucleotides opposite highly distorting or noninstructional DNA lesions. Here, we provide evidence for the physical interaction of hPolι 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 hPolι. In the presence of these protein factors, on undamaged DNA, the efficiency ( V max / K m ) of correct nucleotide incorporation by hPolι is increased ≈80–150-fold, and this increase in efficiency results from a reduction in the apparent K m for the nucleotide. PCNA, RFC, and RPA also stimulate nucleotide incorporation opposite the 3′-T of the (6) thymine–thymine (T-T) photoproduct and opposite an abasic site. The interaction of hPolι with PCNA implies that the targeting of this polymerase to the replication machinery stalled at a lesion site is achieved via this association.

Abasic (AP) sites are one of the most frequently formed lesions in DNA, and they present a strong block to continued synthesis by the replicative DNA machinery. Here we show efficient bypass of an AP site by the combined action of yeast DNA polymerases δ and ζ. In this reaction, Polδ inserts an A nucleotide opposite the AP site, and Polζ subsequently extends from the inserted nucleotide. Consistent with these observations, sequence analyses of mutations in the yeastCAN1s gene indicate that A is the nucleotide inserted most often opposite AP sites. The nucleotides C, G, and T are also incorporated, but much less frequently. Enzymes such as Rev1 and Polη may contribute to the insertion of these other nucleotides; the predominant role of Rev1 in AP bypass, however, is likely to be structural. Steady-state kinetic analyses show that Polζ is highly inefficient in incorporating nucleotides opposite the AP site, but it efficiently extends from nucleotides, particularly an A, inserted opposite this lesion. Thus, in eukaryotes, bypass of an AP site requires the sequential action of two DNA polymerases, wherein the extension step depends solely upon Polζ, but the insertion step can be quite varied, involving not only the predominant action of the replicative DNA polymerase, Polδ, but also the less prominent role of various translesion synthesis polymerases.

After DNA damage, Def1 triggers degradation of the catalytic subunit of the replicative DNA polymerase at stalled replication forks, allowing special polymerases to take over DNA synthesis., DNA damages hinder the advance of replication forks because of the inability of the replicative polymerases to synthesize across most DNA lesions. Because stalled replication forks are prone to undergo DNA breakage and recombination that can lead to chromosomal rearrangements and cell death, cells possess different mechanisms to ensure the continuity of replication on damaged templates. Specialized, translesion synthesis (TLS) polymerases can take over synthesis at DNA damage sites. TLS polymerases synthesize DNA with a high error rate and are responsible for damage-induced mutagenesis, so their activity must be strictly regulated. However, the mechanism that allows their replacement of the replicative polymerase is unknown. Here, using protein complex purification and yeast genetic tools, we identify Def1 as a key factor for damage-induced mutagenesis in yeast. In in vivo experiments we demonstrate that upon DNA damage, Def1 promotes the ubiquitylation and subsequent proteasomal degradation of Pol3, the catalytic subunit of the replicative polymerase δ, whereas Pol31 and Pol32, the other two subunits of polymerase δ, are not affected. We also show that purified Pol31 and Pol32 can form a complex with the TLS polymerase Rev1. Our results imply that TLS polymerases carry out DNA lesion bypass only after the Def1-assisted removal of Pol3 from the stalled replication fork., Author Summary DNA damages can lead to the stalling of the cellular replication machinery if not repaired on time, inducing DNA strand breaks, recombination that can result in gross chromosomal rearrangements, even cell death. In order to guard against this outcome, cells have evolved several precautionary mechanisms. One of these involves the activity of special DNA polymerases—known as translesion synthesis (TLS) polymerases. In contrast to the replicative polymerases responsible for faithfully duplicating the genome, these can carry out DNA synthesis even on a damaged template. For that to occur, they have to take over synthesis from the replicative polymerase that is stalled at a DNA lesion. Although this mechanism allows DNA synthesis to proceed, TLS polymerases work with a high error rate even on undamaged DNA, leading to alterations of the original sequence that can result in cancer. Consequently, the exchange between replicative and special polymerases has to be highly regulated, and the details of this are largely unknown. Here we identified Def1—a protein involved in the degradation of RNA polymerase II—as a prerequisite for error-prone DNA synthesis in yeast. We showed that after treating the cells with a DNA damaging agent, Def1 promoted the degradation of the catalytic subunit of the replicative DNA polymerase δ, without affecting the other two subunits of the polymerase. Our data suggest that the special polymerases can take over synthesis only after the catalytic subunit of the replicative polymerase is removed from the stalled fork in a regulated manner. We predict that the other two subunits remain at the fork and participate in TLS together with the special polymerases.

Summary Lesions in the template DNA strand block the progression of the replication fork. In the yeast Saccharomyces cerevisiae, replication through DNA lesions is mediated by different Rad6-Rad18-dependent means, which include translesion synthesis and a Rad5-dependent postreplicational repair pathway that repairs the discontinuities that form in the DNA synthesized from damaged templates. Although translesion synthesis is well characterized, little is known about the mechanisms that modulate Rad5-dependent postreplicational repair. Here we show that yeast Rad5 has a DNA helicase activity that is specialized for replication fork regression. On model replication fork structures, Rad5 concertedly unwinds and anneals the nascent and the parental strands without exposing extended single-stranded regions. These observations provide insight into the mechanism of postreplicational repair in which Rad5 action promotes template switching for error-free damage bypass.

Human SHPRH gene is located at the 6q24 chromosomal region, and loss of heterozygosity in this region is seen in a wide variety of cancers. SHPRH is a member of the SWI/SNF family of ATPases/helicases, and it possesses a C 3 HC 4 RING motif characteristic of ubiquitin ligase proteins. In both of these features, SHPRH resembles the yeast Rad5 protein, which, together with Mms2–Ubc13, promotes replication through DNA lesions via an error-free postreplicational repair pathway. Genetic evidence in yeast has indicated a role for Rad5 as a ubiquitin ligase in mediating the Mms2–Ubc13-dependent polyubiquitylation of proliferating cell nuclear antigen. Here we show that SHPRH is a functional homolog of Rad5. Similar to Rad5, SHPRH physically interacts with the Rad6–Rad18 and Mms2–Ubc13 complexes, and we show that SHPRH protein is a ubiquitin ligase indispensable for Mms2–Ubc13-dependent polyubiquitylation of proliferating cell nuclear antigen. Based on these observations, we predict a role for SHPRH in promoting error-free replication through DNA lesions. Such a role for SHPRH is consistent with the observation that this gene is mutated in a number of cancer cell lines, including those from melanomas and ovarian cancers, which raises the strong possibility that SHPRH function is an important deterrent to mutagenesis and carcinogenesis in humans.

The Rad6-Rad18 ubiquitin-conjugating enzyme complex of Saccharomyces cerevisiae promotes replication through DNA lesions via three separate pathways that include translesion synthesis (TLS) by DNA polymerases eta and zeta and postreplicational repair (PRR) of discontinuities that form in the newly synthesized DNA opposite from DNA lesions, mediated by the Mms2-Ubc13 ubiquitin-conjugating enzyme and Rad5. Rad5 is an SWI/SNF family ATPase, and additionally, it functions as a ubiquitin ligase in the ubiquitin conjugation reaction. To decipher the roles of these Rad5 activities in lesion bypass, here we examine the effects of mutations in the Rad5 ATPase and ubiquitin ligase domains on the PRR of UV-damaged DNA and on UV-induced mutagenesis. Even though the ATPase-defective mutation confers only a modest degree of UV sensitivity whereas the ubiquitin ligase mutation causes a high degree of UV sensitivity, we find that both of these mutations produce the same high level of PRR defect as that conferred by the highly UV-sensitive rad5Delta mutation. From these studies, we infer a requirement of the Rad5 ATPase and ubiquitin ligase activities in PRR, and based upon the effects of different rad5 mutations on UV mutagenesis, we suggest a role for Rad5 in affecting the efficiency of lesion bypass by the TLS polymerases. In contrast to the role of Rad5 in PRR, however, where its function is coupled with that of Mms2-Ubc13, Rad5 function in TLS would be largely independent of this ubiquitin-conjugating enzyme complex.

The Rad6–Rad18 ubiquitin-conjugating enzyme complex promotes replication through DNA lesions by means of at least three different pathways: the DNA polymerase (Pol) η- and ζ-dependent translesion DNA synthesis (TLS) and a Rad5–Mms2–Ubc13-dependent pathway. In DNA-damaged yeast cells proliferating cell nuclear antigen (PCNA) becomes monoubiquitylated at the K164 residue, and genetic studies in yeast have indicated a requirement for this modification in TLS mediated by Polη and Polζ. To be able to decipher the role of PCNA monoubiquitylation in the TLS process, we have reconstituted this PCNA modification in vitro from purified yeast proteins. We show that, in addition to the requirement for Rad6–Rad18, the reaction depends on the loading of the PCNA homotrimeric ring onto the DNA by replication factor C and that all three PCNA monomers become efficiently ubiquitylated. The availability of PCNA monoubiquitylated on all of its three monomers has enabled us to examine the effects of this PCNA modification on DNA synthesis by Pols δ, η, ζ, and Rev1. Contrary to the prevailing ideas that presume a role for PCNA ubiquitylation in the disruption of Polδ’s binding to PCNA or in the enhancement of the binding affinity of the TLS Pols for PCNA, we find that PCNA ubiquitylation does not affect any of these processes. These observations lead us to suggest a role for PCNA monoubiquitylation in disrupting the PCNA binding of a protein(s) that otherwise is inhibitory to the binding of PCNA by TLS Pols.

The Rev1 protein of Saccharomyces cerevisiae functions in translesion synthesis (TLS) together with DNA polymerase (Pol) zeta, which is comprised of the Rev3 catalytic and the Rev7 accessory subunits. Rev1, a member of the Y family of Pols, differs from other members in its high degree of specificity for incorporating a C opposite template G as well as opposite an abasic site. Although Rev1 is indispensable for Polzeta-dependent TLS, its DNA synthetic activity is not required for many of the Polzeta-dependent lesion bypass events. This observation has suggested a structural role for Rev1 in this process. Here we show that in yeast, Rev1 forms a stable complex with Rev7, and the two proteins copurify. Importantly, the polymerase-associated domain (PAD) of Rev1 mediates its binding to Rev7. These observations reveal a novel role for the PAD region of Rev1 in protein-protein interactions, and they raise the possibility of a similar involvement of the PAD of other Y family Pols in protein-protein interactions. We discuss the possible roles of Rev1 versus the Rev1-Rev7 complex in TLS.

Proliferating cell nuclear antigen (PCNA), a highly conserved, ring-shaped homotrimeric eukaryotic protein, forms a sliding clamp at the template-primer junction. PCNA is loaded onto the primer-template junction in an ATP-dependent manner by a multiprotein clamp loader, replication factor C (RFC). After the loading of PCNA, RFC stays on the DNA via interaction with replication protein A (RPA) bound to single-stranded DNA (1, 18, 37). The binding of the replicative DNA polymerase (Pol), Polδ, to PCNA endows it with a very high processivity (25, 30), and that presumably is the essential function of PCNA in DNA replication. In Saccharomyces cerevisiae, Polδ is comprised of three subunits of 125, 55, and 40 kDa, encoded by the POL3, POL31, and POL32 genes, respectively (6). While the Pol3 catalytic subunit and the Pol31 subunit are highly conserved among eukaryotes, the Pol32 subunit shows a high degree of divergence. The S. cerevisiae Pol3, Pol31, and Pol32 subunits are the respective homologs of Schizosaccharomyces pombe Polδ subunits Pol3, Cdc1, and Cdc27. Whereas the Pol3 and Pol31 subunits and their counterparts are essential in both S. cerevisiae and S. pombe, the third subunit, Cdc27, is essential for completion of the S phase in S. pombe (22, 27), but its counterpart in S. cerevisiae, Pol32, is not. pol32Δ cells, however, grow poorly and exhibit DNA replication defects (6). A series of genetic and biochemical observations with S. cerevisiae Polδ have indicated that at least two separate domains on Polδ interact with at least two separate domains on PCNA, and furthermore, it has been suggested that, during replication, Polδ binds to at least two PCNA monomers (15). Overall, the various studies with Polδ from S. cerevisiae, S. pombe, and humans have strongly indicated that several distinct domains on Polδ interact with different regions of PCNA, and these multiple interactions provide the high degree of processivity that PCNA binding imparts to Polδ. Briefly, we review this evidence below. A consensus PCNA binding motif, QXX(L/I)XXFF, is present at the extreme C terminus of Pol32 in S. cerevisiae and also in its S. pombe and human counterparts Cdc27 and p66, respectively, and mutational inactivation of this domain in PCNA from S. cerevisiae and S. pombe affects the processivity of Polδ (2, 15). In addition, biochemical studies with two mutant PCNAs from S. cerevisiae, pcna-79 and pcna-90, have shown that they both affect the processivity of Polδ (5, 15). In the pcna-79 mutant (I126A/L128A), the hydrophobic pocket in the interdomain connector loop (IDCL) of PCNA is impaired (5), and this mutant PCNA fails to interact with proteins via their consensus QXX(L/I)XXFF PCNA binding motif (8, 15, 32). The pcna-90 (P252A/K253A) mutant has mutational changes in the carboxy-terminal tail of PCNA (5). Since the carboxy-terminal tail of PCNA does not interact with the consensus PCNA binding motif present in Pol32, the adverse effects of mutations in this PCNA region on Polδ processivity (15) must derive from interactions of PCNA with Polδ at a site different from the IDCL interacting domain of Pol32. In keeping with this idea, at least two PCNA binding sites have been identified in the p125 catalytic subunit of human Polδ: one of these is contained in the N2 region toward the amino terminus (38), and the other is in the succeeding N4 region (36). The latter sequence is characterized by the presence of a highly conserved KA motif. The association of Polδ with PCNA thus would be considerably strengthened by these multisite interactions. The Y family DNA Pols, such as Polη, Polι, and Polκ, promote replication through distorting DNA lesions, but they replicate DNA with a low fidelity and low processivity (24). Although all these Pols interact with PCNA, physically as well as functionally, binding to PCNA does not improve their processivity (9-11, 13). For example, PCNA, when loaded onto DNA by RFC in the presence of RPA, stimulates the DNA synthetic activity of both yeast and human Polη approximately 10- to 15-fold, but the processivity in the presence of these protein factors remains the same as in their absence, at about three or four nucleotides per DNA binding event (9, 11). Instead, the increase in the efficiency of nucleotide incorporation is achieved primarily by a reduction in the apparent Km for the nucleotide (9, 11). PCNA, in the presence of RFC and RPA, also greatly stimulates the DNA synthetic activity of Polι and Polκ, and this again is achieved by a decrease in the apparent Km for the nucleotide, whereas the processivity remains unaffected (10, 13). Since PCNA binding does not improve the processivity of Y family DNA Pols, these Pols must differ in their mode of PCNA binding from Polδ. As multisite interactions would provide for the strong association of Polδ with PCNA and the ensuing large increase in Polδ processivity, we have examined the possibility that the lack of PCNA stimulation of the processivity of Y family Pols derives from their binding to PCNA rather weakly. The presence of multiple putative PCNA binding motifs in Polι prompted us to determine whether this Pol made multisite contacts with PCNA or whether only one of the sites was involved. Here we show that Polι interacts with PCNA via only one of these sites and discuss the implications of this observation for translesion DNA synthesis (TLS) by Polι as well as other Y family Pols.

The Apn2 protein of Saccharomyces cerevisiae contains 335 exonuclease and 3-phosphodiesterase activities, and these activities function in the repair of DNA strand breaks that have 3-damaged termini and which are formed in DNA by the action of oxygen-free radicals. Apn2 also has an AP endonuclease activity and functions in the removal of abasic sites from DNA. Here, we provide evidence for the physical and functional interaction of Apn2 with proliferating cell nuclear antigen (PCNA). As indicated by gel filtration and twohybrid studies, Apn2 interacts with PCNA both in vitro and in vivo and mutations in the consensus PCNAbinding motif of Apn2 abolish this interaction. Importantly, PCNA stimulates the 335 exonuclease and 3-phosphodiesterase activities of Apn2. We have examined the involvement of the interdomain connector loop (IDCL) and of the carboxy-terminal domain of PCNA in Apn2 binding and found that Apn2 binds PCNA via distinct domains dependent upon whether the binding is in the absence or presence of DNA. In the absence of DNA, Apn2 binds PCNA through its IDCL domain, whereas in the presence of DNA, when PCNA has been loaded onto the template-primer junction by replication factor C, the C-terminal domain of PCNA mediates the binding. Cellular DNA is continually being damaged by a variety of extrinsic and intrinsic factors. Oxygen-free radicals formed during the course of normal cellular metabolism produce modified miscoding bases, abasic (AP) sites, and DNA strand breaks. AP sites are some of the most common lesions that arise in cellular DNA. It has been estimated that a mammalian cell loses up to 10,000 purines per day spontaneously from its genome (18). AP sites are also formed from the action of DNA glycosylases on modified bases. Oxidative attack on DNA also results in DNA strand breaks that have a 3-phosphate or 3-phosphoglycolate (3-PG) terminus (11, 13). The -lyase activity of some DNA glycosylases also leads to the formation of such modified 3 termini (28). Since these 3-end groups are refractory to DNA synthesis, their removal is an essential early step in the repair of these lesions (3). Class II AP endonucleases are multifunctional enzymes that function in the removal of AP sites as well as 3-blocking termini. Two families of class II AP endonuclease and repair diesterase enzymes have been identified. The endonuclease IV family contains endonuclease IV of Escherichia coli and the Apn1 protein, the major AP endonuclease of Saccharomyces cerevisiae. In addition to AP endonuclease activity, these enzymes possess 3-phosphatase and 3-phosphodiesterase activities that can remove 3-modified termini (22, 28). The exonuclease III (exoIII) family includes exoIII, the major AP endonuclease of E. coli, the Apn2 protein of S. cerevisiae, and Ape1, the dominant AP endonuclease in human cells. exoIII displays, in addition to the AP endonuclease activity, strong 335 exonuclease, 3-phosphodiesterase, and 3-phosphatase activities (28). Human Ape1, however, has a strong AP endonuclease activity but weak 335 exonuclease, 3-phosphodiesterase, and 3-phosphatase activities (23, 31). The Apn2 protein of S. cerevisiae exhibits a weak AP endonuclease activity and strong 335 exonuclease and 3-phosphodiesterase activities that are 30- to 40-fold more active than the AP endonuclease activity (25, 26).

Human DNA polymerase eta (hPoleta) functions in the error-free replication of UV-damaged DNA, and mutations in hPoleta 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 hPoleta is targeted to the replication machinery stalled at a lesion site remains unknown. Here, we provide evidence for the physical interaction of hPoleta with proliferating cell nuclear antigen (PCNA) and show that mutations in the PCNA binding motif of hPoleta inactivate this interaction. PCNA, together with replication factor C and replication protein A, stimulates the DNA synthetic activity of hPoleta, 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.

In both yeast and humans, DNA polymerase (Pol) eta functions in error-free replication of ultraviolet-damaged DNA, and Poleta promotes replication through many other DNA lesions as well. Here, we present evidence for the physical and functional interaction of yeast Poleta with proliferating cell nuclear antigen (PCNA) and show that the interaction with PCNA is essential for the in vivo function of Poleta. Poleta is highly inefficient at inserting a nucleotide opposite an abasic site, but interaction with PCNA greatly stimulates its ability for nucleotide incorporation opposite this lesion. Thus, in addition to having a pivotal role in the targeting of Poleta to the replication machinery stalled at DNA lesions, interaction with PCNA would promote the bypass of certain DNA lesions.

In Saccharomyces cerevisiae, the AP endonucleases encoded by the APN1 and APN2 genes provide alternate pathways for the removal of abasic sites. Oxidative DNA-damaging agents, such as H(2)O(2), produce DNA strand breaks which contain 3'-phosphate or 3'-phosphoglycolate termini. Such 3' termini are inhibitory to synthesis by DNA polymerases. Here, we show that purified yeast Apn2 protein contains 3'-phosphodiesterase and 3'--5' exonuclease activities, and mutation of the active-site residue Glu59 to Ala in Apn2 inactivates both these activities. Consistent with these biochemical observations, genetic studies indicate the involvement of APN2 in the repair of H(2)O(2)-induced DNA damage in a pathway alternate to APN1, and the Ala59 mutation inactivates this function of Apn2. From these results, we conclude that the ability of Apn2 to remove 3'-end groups from DNA is paramount for the repair of strand breaks arising from the reaction of DNA with reactive oxygen species.

Abasic (apurinic/apyrimidinic; AP) sites are generated in vivo through spontaneous base loss and by enzymatic removal of bases damaged by alkylating agents and reactive oxygen species. In Saccharomyces cerevisiae, the APN1 and APN2 genes function in alternate pathways of AP site removal. Apn2-like proteins have been identified in other eukaryotes including humans, and these proteins form a distinct subfamily within the exonuclease III (ExoIII)/Ape1/Apn2 family of proteins. Apn2 and other members of this subfamily contain a carboxyl-terminal extension not present in the ExoIII/Ape1-like proteins. Here, we purify the Apn2 protein from yeast and show that it is a class II AP endonuclease. Deletion of the carboxyl terminus does not affect the AP endonuclease activity of the protein, but this protein is defective in the removal of AP sites in vivo. The carboxyl terminus may enable Apn2 to complex with other proteins, and such a multiprotein assembly may be necessary for the efficient recognition and cleavage of AP sites in vivo.