E3 ubiquitin ligase RNF2 protects polymerase ι from destabilization.

Human DNA polymerase ι (Polι) belongs to the Y-family of specialized DNA polymerases engaged in the DNA damage tolerance pathway of translesion DNA synthesis that is crucial to the maintenance of genome integrity. The extreme infidelity of Polι and the fact that both its up- and down-regulation correlate with various cancers indicate that Polι expression and access to the replication fork should be strictly controlled. Here, we identify RNF2, an E3 ubiquitin ligase, as a new interacting partner of Polι that is responsible for Polι stabilization in vivo. Interestingly, while we report that RNF2 does not directly ubiquitinate Polι, inhibition of the E3 ubiquitin ligase activity of RNF2 affects the cellular level of Polι thereby protecting it from destabilization. Additionally, we indicate that this mechanism is more general, as DNA polymerase η, another Y-family polymerase and the closest paralogue of Polι, share similar features.

A Novel Interaction Between RAD23A/B and Y-family DNA Polymerases.

The Y-family DNA polymerases - Pol ι, Pol η, Pol κ and Rev1 - are most well-known for their roles in the DNA damage tolerance pathway of translesion synthesis (TLS). They function to overcome replication barriers by bypassing DNA damage lesions that cannot be normally replicated, allowing replication forks to continue without stalling. In this work, we demonstrate a novel interaction between each Y-family polymerase and the nucleotide excision repair (NER) proteins, RAD23A and RAD23B. We initially focus on the interaction between RAD23A and Pol ι, and through a series of biochemical, cell-based, and structural assays, find that the RAD23A ubiquitin-binding domains (UBA1 and UBA2) interact with separate sites within the Pol ι catalytic domain. While this interaction involves the ubiquitin-binding cleft of UBA2, Pol ι interacts with a distinct surface on UBA1. We further find that mutating or deleting either UBA domain disrupts the RAD23A-Pol ι interaction, demonstrating that both interactions are necessary for stable binding. We also provide evidence that both RAD23 proteins interact with Pol ι in a similar manner, as well as with each of the Y-family polymerases. These results shed light on the interplay between the different functions of the RAD23 proteins and reveal novel binding partners for the Y-family TLS polymerases.

Regulation of the abundance of Y-family polymerases in the cell cycle of budding yeast in response to DNA damage.

Y-family DNA polymerases mediate DNA damage tolerance via translesion synthesis (TLS). Because of the intrinsically error-prone nature of these enzymes, their activities are regulated at several levels. Here, we demonstrate the common regulation of the cellular abundance of Y-family polymerases, polymerase eta (Pol eta), and Rev1, in response to DNA damage at various stages of the cell cycle. UV radiation influenced polymerase abundance more when cells were exposed in S-phase than in G1- or G2-phases. We noticed two opposing effects of UV radiation in S-phase. On one hand, exposure to increasing doses of UV radiation at the beginning of this phase increasingly delayed S-phase progression. As a result, the accumulation of Pol eta and Rev1, which in nonirradiated yeast is initiated at the S/G2-phase boundary, was gradually shifted into the prolonged S-phase. On the other hand, the extent of polymerase accumulation was inversely proportional to the dose of irradiation, such that the accumulation was significantly lower after exposure to 80 J/m2 in S-phase than after exposure to 50 J/m2 or 10 J/m2. The limitation of polymerase accumulation in S-phase-arrested cells in response to high UV dose was suppressed upon RAD9 (but not MRC1) deletion. Additionally, hydroxyurea, which activates mainly the Mrc1-dependent checkpoint, did not limit Pol eta or Rev1 accumulation in S-phase-arrested cells. The results show that the accumulation of Y-family TLS polymerases is limited in S-phase-arrested cells due to high levels of DNA damage and suggest a role of the Rad9 checkpoint protein in this process.

Polymerase iota - an odd sibling among Y family polymerases.

It has been two decades since the discovery of the most mutagenic human DNA polymerase, polymerase iota (Polι). Since then, the biochemical activity of this translesion synthesis (TLS) enzyme has been extensively explored, mostly through in vitro experiments, with some insight into its cellular activity. Polι is one of four members of the Y-family of polymerases, which are the best characterized DNA damage-tolerant polymerases involved in TLS. Polι shares some common Y-family features, including low catalytic efficiency and processivity, high infidelity, the ability to bypass some DNA lesions, and a deficiency in 3'→5' exonucleolytic proofreading. However, Polι exhibits numerous properties unique among the Y-family enzymes. Polι has an unusual catalytic pocket structure and prefers Hoogsteen over Watson-Crick pairing, and its replication fidelity strongly depends on the template; further, it prefers Mn2+ ions rather than Mg2+ as catalytic activators. In addition to its polymerase activity, Polι possesses also 5'-deoxyribose phosphate (dRP) lyase activity, and its ability to participate in base excision repair has been shown. As a highly error-prone polymerase, its regulation is crucial and mostly involves posttranslational modifications and protein-protein interactions. The upregulation and downregulation of Polι are correlated with different types of cancer and suggestions regarding the possible function of this polymerase have emerged from studies of various cancer lines. Nonetheless, after twenty years of research, the biological function of Polι  certainly remains unresolved.

DNA polymerase ι is acetylated in response to SN2 alkylating agents.

DNA polymerase iota (Polι) belongs to the Y-family of DNA polymerases that are involved in DNA damage tolerance through their role in translesion DNA synthesis. Like all other Y-family polymerases, Polι interacts with proliferating cell nuclear antigen (PCNA), Rev1, ubiquitin and ubiquitinated-PCNA and is also ubiquitinated itself. Here, we report that Polι also interacts with the p300 acetyltransferase and is acetylated. The primary acetylation site is K550, located in the Rev1-interacting region. However, K550 amino acid substitutions have no effect on Polι's ability to interact with Rev1. Interestingly, we find that acetylation of Polι significantly and specifically increases in response to SN2 alkylating agents and to a lower extent to SN1 alkylating and oxidative agents. As we have not observed acetylation of Polι's closest paralogue, DNA polymerase eta (Polη), with which Polι shares many functional similarities, we believe that this modification might exclusively regulate yet to be determined, and separate function(s) of Polι.

Posttranslational Regulation of Human DNA Polymerase ι.

Human DNA polymerases (pols) η and ι are Y-family DNA polymerase paralogs that facilitate translesion synthesis past damaged DNA. Both polη and polι can be monoubiquitinated in vivo. Polη has been shown to be ubiquitinated at one primary site. When this site is unavailable, three nearby lysines may become ubiquitinated. In contrast, mass spectrometry analysis of monoubiquitinated polι revealed that it is ubiquitinated at over 27 unique sites. Many of these sites are localized in different functional domains of the protein, including the catalytic polymerase domain, the proliferating cell nuclear antigen-interacting region, the Rev1-interacting region, and its ubiquitin binding motifs UBM1 and UBM2. Polι monoubiquitination remains unchanged after cells are exposed to DNA-damaging agents such as UV light (generating UV photoproducts), ethyl methanesulfonate (generating alkylation damage), mitomycin C (generating interstrand cross-links), or potassium bromate (generating direct oxidative DNA damage). However, when exposed to naphthoquinones, such as menadione and plumbagin, which cause indirect oxidative damage through mitochondrial dysfunction, polι becomes transiently polyubiquitinated via Lys(11)- and Lys(48)-linked chains of ubiquitin and subsequently targeted for degradation. Polyubiquitination does not occur as a direct result of the perturbation of the redox cycle as no polyubiquitination was observed after treatment with rotenone or antimycin A, which both inhibit mitochondrial electron transport. Interestingly, polyubiquitination was observed after the inhibition of the lysine acetyltransferase KATB3/p300. We hypothesize that the formation of polyubiquitination chains attached to polι occurs via the interplay between lysine acetylation and ubiquitination of ubiquitin itself at Lys(11) and Lys(48) rather than oxidative damage per se.

Regulation of translesion DNA synthesis: Posttranslational modification of lysine residues in key proteins.

Posttranslational modification of proteins often controls various aspects of their cellular function. Indeed, over the past decade or so, it has been discovered that posttranslational modification of lysine residues plays a major role in regulating translesion DNA synthesis (TLS) and perhaps the most appreciated lysine modification is that of ubiquitination. Much of the recent interest in ubiquitination stems from the fact that proliferating cell nuclear antigen (PCNA) was previously shown to be specifically ubiquitinated at K164 and that such ubiquitination plays a key role in regulating TLS. In addition, TLS polymerases themselves are now known to be ubiquitinated. In the case of human polymerase η, ubiquitination at four lysine residues in its C-terminus appears to regulate its ability to interact with PCNA and modulate TLS. Within the past few years, advances in global proteomic research have revealed that many proteins involved in TLS are, in fact, subject to a previously underappreciated number of lysine modifications. In this review, we will summarize the known lysine modifications of several key proteins involved in TLS; PCNA and Y-family polymerases η, ι, κ and Rev1 and we will discuss the potential regulatory effects of such modification in controlling TLS in vivo.

The steady-state level and stability of TLS polymerase eta are cell cycle dependent in the yeast S. cerevisiae.

Polymerase eta (Pol eta) is a ubiquitous translesion DNA polymerase that is capable of bypassing UV-induced pyrimidine dimers in an error-free manner. However, this specialized polymerase is error prone when synthesizing through an undamaged DNA template. In Saccharomyces cerevisiae, both depletion and overproduction of Pol eta result in mutator phenotypes. Therefore, regulation of the cellular abundance of this enzyme is of particular interest. However, based on the investigation of variously tagged forms of Pol eta, mutually contradictory conclusions have been reached regarding the stability of this polymerase in yeast. Here, we optimized a protocol for the detection of untagged yeast Pol eta and established that the half-life of the native enzyme is 80 ± 14 min in asynchronously growing cultures. Experiments with synchronized cells indicated that the cellular abundance of this translesion polymerase changes throughout the cell cycle. Accordingly, we show that the stability of Pol eta, but not its mRNA level, is cell cycle stage dependent. The half-life of the polymerase is more than fourfold shorter in G1-arrested cells than in those at G2/M. Our results, in concert with previous data for Rev1, indicate that cell cycle regulation is a general property of Y family TLS polymerases in S. cerevisiae.

Ubiquitin mediates the physical and functional interaction between human DNA polymerases η and ι.

Human DNA polymerases η and ι are best characterized for their ability to facilitate translesion DNA synthesis (TLS). Both polymerases (pols) co-localize in 'replication factories' in vivo after cells are exposed to ultraviolet light and this co-localization is mediated through a physical interaction between the two TLS pols. We have mapped the polη-ι interacting region to their respective ubiquitin-binding domains (UBZ in polη and UBM1 and UBM2 in polι), and demonstrate that ubiquitination of either TLS polymerase is a prerequisite for their physical and functional interaction. Importantly, while monoubiquitination of polη precludes its ability to interact with proliferating cell nuclear antigen (PCNA), it enhances its interaction with polι. Furthermore, a polι-ubiquitin chimera interacts avidly with both polη and PCNA. Thus, the ubiquitination status of polη, or polι plays a key regulatory function in controlling the protein partners with which each polymerase interacts, and in doing so, determines the efficiency of targeting the respective polymerase to stalled replication forks where they facilitate TLS.

The roles of PCNA SUMOylation, Mms2-Ubc13 and Rad5 in translesion DNA synthesis in Saccharomyces cerevisiae.

Mms2, in concert with Ubc13 and Rad5, is responsible for polyubiquitination of replication processivity factor PCNA. This modification activates recombination-like DNA damage-avoidance mechanisms, which function in an error-free manner. Cells deprived of Mms2, Ubc13 or Rad5 exhibit mutator phenotypes as a result of the channelling of premutational DNA lesions to often error-prone translesion DNA synthesis (TLS). Here we show that Siz1-mediated PCNA SUMOylation is required for the stimulation of this TLS, despite the presence of PCNA monoubiquitination. The stimulation of spontaneous mutagenesis by Siz1 in cells carrying rad5 and/or mms2 mutations is connected with the known role of PCNA SUMOylation in the inhibition of Rad52-mediated recombination. However, following UV irradiation, Siz1 is engaged in additional, as yet undefined, mechanisms controlling genetic stability at the replication fork. We also demonstrate that in the absence of PCNA SUMOylation, Mms2-Ubc13 and Rad5 may, independently of each other, function in the stimulation of TLS. Based on this finding and on an analysis of the epistatic relationships between SIZ1, MMS2 and RAD5, with respect to UV sensitivity, we conclude that PCNA SUMOylation is responsible for the functional differences between the Mms2 and Rad5 homologues of Saccharomyces cerevisiae and Schizosaccharomyces pombe.

The spectrum of spontaneous mutations caused by deficiency in proteasome maturase Ump1 in Saccharomyces cerevisiae.

Ump1 is responsible for maturation of the catalytic core of the 26S proteasome. Dysfunction of Ump1 causes an increase in the frequency of spontaneous mutations in Saccharomyces cerevisiae. In this study we analyze the spectrum of mutations occurring spontaneously in yeast deficient in Ump1 by use of the SUP4-o system. Single base substitutions predominate among the mutations analyzed (73 of the 91 alterations examined). Two major classes are GC to TA transversions and GC to AT transitions ( approximately 50 and approximately 30% of base substitutions, respectively). Besides base substitutions, almost all the major types of sequence alterations are represented. The specificity and distribution of mutations occurring in the ump1 strain are unique compared to the spectra previously established for other yeast mutators. However, the profile of mutations arising in this strain is similar to that observed in wild type. The same similarity has previously been reported for yeast deficient in Mms2, a protein involved in Rad6-dependent postreplication DNA repair (PRR). The specificity of the mutator effect caused by ump1 is discussed in light of the proposed role of the proteasome activity in the regulation of the PRR mechanisms.

Polymerase eta is a short-lived, proteasomally degraded protein that is temporarily stabilized following UV irradiation in Saccharomyces cerevisiae.

Saccharomyces cerevisiae Rad30 is the homolog of human DNA polymerase eta whose inactivation leads to the cancer-prone syndrome xeroderma pigmentosum variant. Both human and yeast polymerase eta are responsible for error-free bypass of UV-induced cis-syn pyrimidine dimers and several other DNA lesions. Here we show, using yeast strains expressing TAP-tagged Rad30, that the level of this protein is post-translationally regulated via ubiquitination and proteasome-mediated degradation. The half-life of Rad30 is 20 min and it increases due to proteasomal defects. Mutations inactivating components of the Skp1/cullin/ F-box (SCF) ubiquitin ligase complex: Skp1 and the F-box protein Ufo1 stabilize Rad30. Our results indicate also that ultraviolet irradiation causes transient stabilization of Rad30, which leads, in turn, to temporary accumulation of this polymerase in the cell. We conclude that proteolysis plays an important role in regulating the cellular abundance of Rad30. These results are the first indication of a role for controlled proteasomal degradation in modulating cellular level of translesion DNA polymerase in eukaryotes.

Analysis of the spontaneous mutator phenotype associated with 20S proteasome deficiency in S. cerevisiae.

Besides its role as a major recycler of unfolded or otherwise damaged intracellular proteins, the 26S proteasome functions as a regulator of many vital cellular processes and is postulated as a target for antitumor drugs. It has previously been shown that dysfunction of the catalytic core of the 26S proteasome, the 20S proteasome, causes a moderate increase in the frequency of spontaneous mutations in yeast [A. Podlaska, J. McIntyre, A. Skoneczna, E. Sledziewska-Gojska, The link between proteasome activity and postreplication DNA repair in Saccharomyces cerevisiae. Mol. Microbiol. 49 (2003) 1321-1332]. Here we show the results of genetic analysis, which indicate that the mutator phenotype caused by the deletion of UMP1, encoding maturase of 20S proteasome, involves members of the RAD6 epistasis group. The great majority of mutations occurring spontaneously in yeast cells deficient in 20S proteasome function are connected with the unique Rad6/Rad18-dependent error-prone translesion DNA synthesis (TLS) requiring the activities of both TLS polymerases: Pol eta and Pol zeta. Our results suggest the involvement of proteasomal activity in the limitation of this unique error-prone TLS mechanism in wild-type cells. On the other hand, we found that the mutator phenotypes caused by deficiency in Rad18 and Rad6, are largely alleviated by defects in proteasome activities. Since the mutator phenotypes produced by deletion of RAD6 and RAD18 require Pol zeta and Siz1/Ubc9-dependent sumoylation of PCNA, our results suggest that proteasomal dysfunction limits sumoylation-dependent error-prone activity of Pol zeta. Taken together, our findings strongly support the idea that proteolytic activity is involved in modulating the balance between TLS mechanisms functioning during DNA replication in S. cerevisiae.

The link between 20S proteasome activity and post-replication DNA repair in Saccharomyces cerevisiae.

We have shown previously that deletion of the Saccharomyces cerevisiae UMP1 gene encoding the 20S proteasome maturase causes sensitivity to UV radiation. In the current report, we have extended this finding to show that mutations specifically compromising chymotrypsin-like or trypsin-like activity of 20S proteasome peptidases also result in increased UV sensitivity. We have also established that mutations affecting proteasome activity, namely ump1Delta, pre2-K108R and pup1-T20A, result in spontaneous and UV-induced mutator phenotypes. To elucidate the origin of these DNA repair phenotypes of the proteasomal mutants, we performed epistasis analysis, with respect to UV sensitivity, using yeast strains with the UMP1 deletion in different DNA repair backgrounds. We show that UMP1 is not epistatic to RAD23 and RAD2, which are involved in the nucleotide excision repair (NER) pathway. Instead, our results indicate that UMP1 as well as PUP1 and PRE2 (encoding catalytic subunits of 20S proteasome) belong to an epistatic group of genes functioning in post-replication DNA repair (PRR) and are hypostatic to RAD18, which, in complex with RAD6, plays a central role in PRR. We also show that UMP1 is epistatic to REV3 and RAD30, although the relationship of UMP1 with these genes is different.