For the Better or for the Worse? The Effect of Manganese on the Activity of Eukaryotic DNA Polymerases.

DNA polymerases constitute a versatile group of enzymes that not only perform the essential task of genome duplication but also participate in various genome maintenance pathways, such as base and nucleotide excision repair, non-homologous end-joining, homologous recombination, and translesion synthesis. Polymerases catalyze DNA synthesis via the stepwise addition of deoxynucleoside monophosphates to the 3' primer end in a partially double-stranded DNA. They require divalent metal cations coordinated by active site residues of the polymerase. Mg2+ is considered the likely physiological activator because of its high cellular concentration and ability to activate DNA polymerases universally. Mn2+ can also activate the known DNA polymerases, but in most cases, it causes a significant decrease in fidelity and/or processivity. Hence, Mn2+ has been considered mutagenic and irrelevant during normal cellular function. Intriguingly, a growing body of evidence indicates that Mn2+ can positively influence some DNA polymerases by conferring translesion synthesis activity or altering the substrate specificity. Here, we review the relevant literature focusing on the impact of Mn2+ on the biochemical activity of a selected set of polymerases, namely, Polß, Polλ, and Polµ, of the X family, as well as Polι and Polη of the Y family of polymerases, where congruous data implicate the physiological relevance of Mn2+ in the cellular function of these enzymes.

The inner side of yeast PCNA contributes to genome stability by mediating interactions with Rad18 and the replicative DNA polymerase δ.

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.

The Rad5 Helicase and RING Domains Contribute to Genome Stability through their Independent Catalytic Activities.

Genomic stability is compromised by DNA damage that obstructs replication. Rad5 plays a prominent role in DNA damage bypass processes that evolved to ensure the continuation of stalled replication. Like its human orthologs, the HLTF and SHPRH tumor suppressors, yeast Rad5 has a RING domain that supports ubiquitin ligase activity promoting PCNA polyubiquitylation and a helicase domain that in the case of HLTF and Rad5 was shown to exhibit an ATPase-linked replication fork reversal activity. The RING domain is embedded in the helicase domain, confusing their separate investigation and the understanding of the exact role of Rad5 in DNA damage bypass. Particularly, it is still debated whether the helicase domain plays a catalytic or a non-enzymatic role during error-free damage bypass and whether it facilitates a function separately from the RING domain. In this study, through in vivo and in vitro characterization of domain-specific mutants, we delineate the contributions of the two domains to Rad5 function. Yeast genetic experiments and whole-genome sequencing complemented with biochemical assays demonstrate that the ubiquitin ligase and the ATPase-linked activities of Rad5 exhibit independent catalytic activities in facilitating separate pathways during error-free lesion bypass. Our results also provide important insights into the mutagenic role of Rad5 and indicate its tripartite contribution to DNA damage tolerance.

The Zn-finger of Saccharomyces cerevisiae Rad18 and its adjacent region mediate interaction with Rad5.

DNA damages that hinder the movement of the replication complex can ultimately lead to cell death. To avoid that, cells possess several DNA damage bypass mechanisms. The Rad18 ubiquitin ligase controls error-free and mutagenic pathways that help the replication complex to bypass DNA lesions by monoubiquitylating PCNA at stalled replication forks. In Saccharomyces cerevisiae, two of the Rad18 governed pathways are activated by monoubiquitylated PCNA and they involve translesion synthesis polymerases, whereas a third pathway needs subsequent polyubiquitylation of the same PCNA residue by another ubiquitin ligase the Rad5 protein, and it employs template switching. The goal of this study was to dissect the regulatory role of the multidomain Rad18 in DNA damage bypass using a structure-function based approach. Investigating deletion and point mutant RAD18 variants in yeast genetic and yeast two-hybrid assays we show that the Zn-finger of Rad18 mediates its interaction with Rad5, and the N-terminal adjacent region is also necessary for Rad5 binding. Moreover, results of the yeast two-hybrid and in vivo ubiquitylation experiments raise the possibility that direct interaction between Rad18 and Rad5 might not be necessary for the function of the Rad5 dependent pathway. The presented data also reveal that yeast Rad18 uses different domains to mediate its association with itself and with Rad5. Our results contribute to better understanding of the complex machinery of DNA damage bypass pathways.

Manganese Is a Strong Specific Activator of the RNA Synthetic Activity of Human Polη.

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.

Selective Metal Ion Utilization Contributes to the Transformation of the Activity of Yeast Polymerase η from DNA Polymerization toward RNA Polymerization.

Polymerase eta (Polη) 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 Polη in transcription elongation, and identified its specific RNA extension and translesion RNA synthetic activities. However, RNA synthesis by Polη 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 Polη. Kinetics experiments indicate that manganese increases the efficiency of ribonucleoside incorporation into RNA by ~400-2000-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 Polη with manganese. Moreover, Polη 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 Polη helping it to perform distinct activities needed for its separate functions during replication and transcription.

Translesion synthesis DNA polymerase η exhibits a specific RNA extension activity and a transcription-associated function.

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.

Mutations at the Subunit Interface of Yeast Proliferating Cell Nuclear Antigen Reveal a Versatile Regulatory Domain.

Proliferating cell nuclear antigen (PCNA) plays a key role in many cellular processes and due to that it interacts with a plethora of proteins. The main interacting surfaces of Saccharomyces cerevisiae PCNA have been mapped to the interdomain connecting loop and to the carboxy-terminal domain. Here we report that the subunit interface of yeast PCNA also has regulatory roles in the function of several DNA damage response pathways. Using site-directed mutagenesis we engineered mutations at both sides of the interface and investigated the effect of these alleles on DNA damage response. Genetic experiments with strains bearing the mutant alleles revealed that mutagenic translesion synthesis, nucleotide excision repair, and homologous recombination are all regulated through residues at the subunit interface. Moreover, genetic characterization of one of our mutants identifies a new sub-branch of nucleotide excision repair. Based on these results we conclude that residues at the subunit boundary of PCNA are not only important for the formation of the trimer structure of PCNA, but they constitute a regulatory protein domain that mediates different DNA damage response pathways, as well.

Def1 promotes the degradation of Pol3 for polymerase exchange to occur during DNA-damage--induced mutagenesis in Saccharomyces cerevisiae.

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.

Measuring UV-induced Mutagenesis at the CAN1 Locus in Saccharomyces cerevisiae.

There are several methods to measure the capacity of yeast cell to respond to environmental impacts on their genome by mutating it. One frequently used method involves the detection of forward mutations in the CAN1 gene. The CAN1 gene encodes for an arginine permease that is responsible for the uptake of arginine and it can also transport the toxic analog of arginine, canavanine (Whelan et al., 1979). When CAN1 cells are grown on a media containing canavanine but lacking arginine, the cells die because of the uptake of the toxic canavanine. However, if a mutation in the CAN1 gene inactivates the permease, that cell survives and forms a colony on the plate. The following protocol describes the measurement of UV-induced mutagenesis at the CAN1 locus.

Synchronization of Saccharomyces cerevisiae Cells in G1 Phase of the Cell Cycle.

The baker's yeast, Saccharomyces cerevisiae is a widely used model organism in molecular biology because of the high homology it shares with human cells in many basic cellular processes such as DNA replication, repair, recombination, transcription, and because of the ease its genome can be manipulated. Other advantages of working with yeast are its fast production rate which is comparable to bacteria's, and its cheap maintenance. To examine certain phenomena, for example whether a mutation affects the passage through a cell cycle phase, it can be necessary to work with a yeast culture, in which all the cells are in the same phase of the cell cycle. Yeasts can be arrested and kept in different phases of the cell cycle. Here we describe the method that allows synchronizing and keeping yeast cells in the G1 phase of the cell cycle with the mating pheromone, α-factor. Only MATa cells can be synchronized with α-factor which is produced by MATα cells. It is highly recommended to use a MATa bar1 deletion strain. The BAR1 gene encodes for an extracellular protease that inactivates α-factor by cleaving it (MacKay et al., 1988). To counteract the Bar1 protease activity when using BAR1 cells, 100-1,000 times more α-factor is needed as compared to bar1 deletion cells (α-factor is quite expensive!), and still the synchrony will be transient. In contrast, bar1 deletion cells can be kept in G1 phase with α-factor for several hours, and the degree of synchronization is usually higher than using a BAR1 strain. Moreover, bar1 deletion cells can be synchronized even at high cell density, whereas BAR1 cells, due to the activity of the secreted Bar1 protease, only at low cell density.

Role of yeast Rad5 and its human orthologs, HLTF and SHPRH in DNA damage tolerance.

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.

Role of double-stranded DNA translocase activity of human HLTF in replication of damaged DNA.

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.

Role of PCNA-dependent stimulation of 3'-phosphodiesterase and 3'-5' exonuclease activities of human Ape2 in repair of oxidative DNA damage.

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.

Roles of PCNA-binding and ubiquitin-binding domains in human DNA polymerase eta in translesion DNA 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 Poleta 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 Poleta'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.

Human HLTF functions as a ubiquitin ligase for proliferating cell nuclear antigen polyubiquitination.

Human helicase-like transcription factor (HLTF) is frequently inactivated in colorectal and gastric cancers. Here, we show that HLTF is a functional homologue of yeast Rad5 that promotes error-free replication through DNA lesions. HLTF and Rad5 share the same unique structural features, including a RING domain embedded within a SWI/SNF helicase domain and an HIRAN domain. We find that inactivation of HLTF renders human cells sensitive to UV and other DNA-damaging agents and that HLTF complements the UV sensitivity of a rad5Delta yeast strain. Also, similar to Rad5, HLTF physically interacts with the Rad6-Rad18 and Mms2-Ubc13 ubiquitin-conjugating enzyme complexes and promotes the Lys-63-linked polyubiquitination of proliferating cell nuclear antigen at its Lys-164 residue. A requirement of HLTF for error-free postreplication repair of damaged DNA is in keeping with its cancer-suppression role.

Yeast Rad5 protein required for postreplication repair has a DNA helicase activity specific for replication fork regression.

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 is a ubiquitin ligase for Mms2-Ubc13-dependent polyubiquitylation of proliferating cell nuclear antigen.

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.

Mms2-Ubc13-dependent and -independent roles of Rad5 ubiquitin ligase in postreplication repair and translesion DNA synthesis in Saccharomyces cerevisiae.

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.

Human Ape2 protein has a 3'-5' exonuclease activity that acts preferentially on mismatched base pairs.

DNA damage, such as abasic sites and DNA strand breaks with 3'-phosphate and 3'-phosphoglycolate termini present cytotoxic and mutagenic threats to the cell. Class II AP endonucleases play a major role in the repair of abasic sites as well as of 3'-modified termini. Human cells contain two class II AP endonucleases, the Ape1 and Ape2 proteins. Ape1 possesses a strong AP-endonuclease activity and weak 3'-phosphodiesterase and 3'-5' exonuclease activities, and it is considered to be the major AP endonuclease in human cells. Much less is known about Ape2, but its importance is emphasized by the growth retardation and dyshematopoiesis accompanied by G2/M arrest phenotype of the APE2-null mice. Here, we describe the biochemical characteristics of human Ape2. We find that Ape2 exhibits strong 3'-5' exonuclease and 3'-phosphodiesterase activities and has only a very weak AP-endonuclease activity. Mutation of the active-site residue Asp 277 to Ala in Ape2 inactivates all these activities. We also demonstrate that Ape2 preferentially acts at mismatched deoxyribonucleotides at the recessed 3'-termini of a partial DNA duplex. Based on these results we suggest a novel role for human Ape2 as a 3'-5' exonuclease.