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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.

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.

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.

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 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).