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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.

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Apurinic/apyrimidic (AP) sites are severe DNA damages and strongly block DNA extension by major DNA polymerases. Y-family DNA polymerases possess a strong ability to bypass AP sites and continue the DNA synthesis reaction, which is called translesion synthesis (TLS) activity. To investigate the effect of the molecular structure of the AP site on the TLS efficiency of Dbh, a Y-family DNA polymerase from Sulfolobus acidocaldarius, a series of different AP site analogues (various spacers) are used to characterize the bypass efficiency. We find that not only the molecular structure and atomic composition but also the number and position of AP site analogues determine the TLS efficiency of Dbh. Increasing the spacer length decreases TLS activity. The TLS efficiency also decreases when more than one spacer exists on the DNA template. The position of the AP site analogues is also an important factor for TLS. When the spacer is opposite to the first incorporated dNTPs, the TLS efficiency is the lowest, suggesting that AP sites are largely harmful for the formation of hydrogen bonds. These results deepen our understanding of the TLS activity of Y-family DNA polymerases and provide a biochemical basis for elucidating the TLS mechanism in Sulfolobus acidocaldarius cells.

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A major mitochondrial enzyme for protecting cells from acetaldehyde toxicity is aldehyde dehydrogenase 2 (ALDH2). The correlation between ALDH2 dysfunction and tumorigenesis/growth/metastasis has been widely reported. Either low or high ALDH2 expression contributes to tumor progression and varies among different tumor types. Furthermore, the ALDH2∗2 polymorphism (rs671) is the most common single nucleotide polymorphism (SNP) in Asia. Epidemiological studies associate ALDH2∗2 with tumorigenesis and progression. This study summarizes the essential functions and potential ALDH2 mechanisms in the occurrence, progression, and treatment of tumors in various types of cancer. Our study indicates that ALDH2 is a potential therapeutic target for cancer therapy.

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With the publication of the first paper describing the biochemical properties of DNA polymerase iota (polɩ), the question immediately arose as to why cells harbor such a low-fidelity enzyme which often violates the Watson-Crick base pairing rules? Yet 20 years after its discovery, the cellular function of polɩ remains unknown. Here, we provide a graphical review of the unique biochemical properties of polɩ and speculate about the cellular pathways in which enigmatic polɩ may participate.

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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.

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Dpo4 is a representative model of Y-family DNA polymerase and is therefore one of the most intensively studied DNA polymerase. 6 mA, an epigenetic marker, plays important roles in regulation of various biological processes. However, its effects on DNA replication by Dpo4 is completely unknown. Here, we found that 6 mA and its intermediate Hyp inhibits primer extension by Dpo4, showing an obvious blockage just one nucleotide before 6 mA or Hyp. 6 mA reduces dTTP incorporation efficiency, next-base extension efficiency, binding affinity of DNA to Dpo4, binding affinity of dTTP to Dpo4-DNA complex, the fraction of productive Dpo4 or productive ternary complex, and the burst incorporation rate, explaining the inhibition effects of 6 mA on DNA replication by Dpo4. Hyp is similar to G and dCTP is preferentially incorporated opposite Hyp by Dpo4, resulting in A:T to G:C mutation. Relative to dTTP incorporation opposite unmodified A, Hyp reduces dCTP incorporation efficiency, next-base extension efficiency, the priority in extension beyond correct pair, binding affinity of Dpo4 to DNA, binding of dCTP to Dpo4-DNA complex, and the burst incorporation efficiency, explaining the inhibition effects of Hyp on DNA replication by Dpo4. This work provides insight in the effects of epigenetically modified 6 mA and Hyp on DNA replication by a representative Y-family DNA polymerase Dpo4.

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pol VICE391 (RumA'2B) is a low-fidelity polymerase that promotes considerably higher levels of spontaneous "SOS-induced" mutagenesis than the related E. coli pol V (UmuD'2C). The molecular basis for the enhanced mutagenesis was previously unknown. Using single molecule fluorescence microscopy to visualize pol V enzymes, we discovered that the elevated levels of mutagenesis are likely due, in part, to prolonged binding of RumB to genomic DNA leading to increased levels of DNA synthesis compared to UmuC. We have generated a steric gate pol VICE391 variant (pol VICE391_Y13A) that readily misincorporates ribonucleotides into the E. coli genome and have used the enzyme to investigate the molecular mechanisms of Ribonucleotide Excision Repair (RER) under conditions of increased ribonucleotide-induced stress. To do so, we compared the extent of spontaneous mutagenesis promoted by pol V and pol VICE391 to that of their respective steric gate variants. Levels of mutagenesis promoted by the steric gate variants that are lower than that of the wild-type enzyme are indicative of active RER that removes misincorporated ribonucleotides, but also misincorporated deoxyribonucleotides from the genome. Using such an approach, we confirmed that RNase HII plays a pivotal role in RER. In the absence of RNase HII, Nucleotide Excision Repair (NER) proteins help remove misincorporated ribonucleotides. However, significant RER occurs in the absence of RNase HII and NER. Most of the RNase HII and NER-independent RER occurs on the lagging strand during genome duplication. We suggest that this is most likely due to efficient RNase HI-dependent RER which recognizes the polyribonucleotide tracts generated by pol VICE391_Y13A. These activities are critical for the maintenance of genomic integrity when RNase HII is overwhelmed, or inactivated, as ΔrnhB or ΔrnhB ΔuvrA strains expressing pol VICE391_Y13A exhibit genome and plasmid instability in the absence of RNase HI.

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DNA polymerases play a key role in mutagenesis by performing translesion DNA synthesis (TLS). The Y-family of DNA polymerases comprises several evolutionarily conserved families, specializing in TLS of different DNA adducts. Exocyclic etheno and propano DNA adducts are among the most common endogenous DNA lesions induced by lipid peroxidation reactions triggered by oxidative stress. We have investigated the participation of two enterobacterial representatives of the PolIV and PolV branches of Y-family DNA polymerases in mutagenesis by two model lipid peroxidation derived genotoxins, glyoxal and crotonaldehyde. Mutagenesis by the ethano adduct (glyoxal-derived) and the propano adduct (crontonaldehyde-derived) at the GC target in the Ames test depended exclusively on PolV type DNA polymerases such as PolRI. In contrast, PolIV suppressed glyoxal and, even more, crotonaldehyde mutagenesis, as detected by enzyme overexpression and gene knockout approaches. We propose that DNA polymerase IV, which is the mammalian DNA polymerase κ ortholog, acts as a housekeeper protecting the genome from lipoxidative stress.

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The cDNA encoding human DNA polymerase ι (POLI) was cloned in 1999. At that time, it was believed that the POLI gene encoded a protein of 715 amino acids. Advances in DNA sequencing technologies led to the realization that there is an upstream, in-frame initiation codon that would encode a DNA polymerase ι (polι) protein of 740 amino acids. The extra 25 amino acid region is rich in acidic residues (11/25) and is reasonably conserved in eukaryotes ranging from fish to humans. As a consequence, the curated Reference Sequence (RefSeq) database identified polι as a 740 amino acid protein. However, the existence of the 740 amino acid polι has never been shown experimentally. Using highly specific antibodies to the 25 N-terminal amino acids of polι, we were unable to detect the longer 740 amino acid (ι-long) isoform in western blots. However, trace amounts of the ι-long isoform were detected after enrichment by immunoprecipitation. One might argue that the longer isoform may have a distinct biological function, if it exhibits significant differences in its enzymatic properties from the shorter, well-characterized 715 amino acid polι. We therefore purified and characterized recombinant full-length (740 amino acid) polι-long and compared it to full-length (715 amino acid) polι-short in vitro. The metal ion requirements for optimal catalytic activity differ slightly between ι-long and ι-short, but under optimal conditions, both isoforms exhibit indistinguishable enzymatic properties in vitro. We also report that like ι-short, the ι-long isoform can be monoubiquitinated and polyubiuquitinated in vivo, as well as form damage induced foci in vivo. We conclude that the predominant isoform of DNA polι in human cells is the shorter 715 amino acid protein and that if, or when, expressed, the longer 740 amino acid isoform has identical properties to the considerably more abundant shorter isoform.

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In 2003, we reported that 129-derived strains of mice carry a naturally occurring nonsense mutation at codon 27 of the Poli gene that would produce a polι peptide of just 26 amino acids, rather then the full-length 717 amino acid wild-type polymerase. In support of the genomic analysis, no polι protein was detected in testes extracts from 129X1/SvJmice, where wild-type polι is normally highly expressed. The early truncation in polι occurs before any structural domains of the polymerase are synthesized and as a consequence, we reasoned that 129-derived strains of mice should be considered as functionally defective in polι activity. However, it has recently been reported that during the maturation of the Poli mRNA in 129-derived strains, exon- 2 is sometimes skipped and that an exon-2-less polι protein of 675 amino acids is synthesized that retains catalytic activity in vitro and in vivo. From a structural perspective, we found this idea untenable, given that the amino acids encoded by exon-2 include residues critical for the coordination of the metal ions required for catalysis, as well as the structural integrity of the DNA polymerase. To determine if the exon-2-less polι isoform possesses catalytic activity in vitro, we have purified a glutathione-tagged full-length exon-2-less (675 amino acid) polι protein from baculovirus infected insect cells and compared the activity of the isoform to full-length (717 amino acid) GST-tagged wild-type mouse polι in vitro. Reaction conditions were performed under a range of magnesium or manganese concentrations, as well as different template sequence contexts. Wild-type mouse polι exhibited robust characteristic properties previously associated with human polι's biochemical properties. However, we did not detect any polymerase activity associated with the exon-2-less polι enzyme under the same reaction conditions and conclude that exon-2-less polι is indeed rendered catalytically inactive in vitro.

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Sulfolobus solfataricus DNA polymerase IV (Dpo4), a model Y-family DNA polymerase, bypasses a wide range of DNA lesions in vitro and in vivo. In this paper, we report the backbone chemical shift assignments of the full length Dpo4 in its binary complex with a 14/14-mer DNA substrate. Upon DNA binding, several ß-stranded regions in the isolated catalytic core and little finger/linker fragments of Dpo4 become more structured. This work serves as a foundation for our ongoing investigation of conformational dynamics of Dpo4 and future determination of the first solution structures of a DNA polymerase and its binary and ternary complexes.

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It is now close to 40 years since the isolation of non-mutable umu/uvm strains of Escherichia coli and the realization that damage induced mutagenesis in E.coli is not a passive process. Early models of mutagenesis envisioned the Umu proteins as accessory factors to the cell's replicase that not only reduced its normally high fidelity, but also allowed the enzyme to traverse otherwise replication-blocking lesions in the genome. However, these models underwent a radical revision approximately 15 years ago, with the discovery that the Umu proteins actually encode for a DNA polymerase, E.coli pol V. The polymerase lacks 3'→5' exonucleolytic proofreading activity and is inherently error-prone when replicating both undamaged and damage DNA. So as to limit any "gratuitous" mutagenesis, the activity of pol V is strictly regulated in the cell at multiple levels. This review will summarize our current understanding of the myriad levels of regulation imposed on pol V including transcriptional control, posttranslational modification, targeted proteolysis, activation of the catalytic activity of pol V through protein-protein interactions and the very recently described intracellular spatial regulation of pol V. Remarkably, despite the multiple levels at which pol V is regulated, the enzyme is nevertheless able to contribute to the genetic diversity and evolutionary fitness of E.coli.

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DNA polymerase κ (Polκ), one of the typical member of the Y-family DNA polymerases, has been demonstrated to bypass the 10S(+)-trans-anti-benzo[a]pyrene diol epoxide-N(2)-deoxyguanine adducts (BPDE-dG) efficiently and accurately. A large structural gap between the core and little finger as well as an N-clasp domain are essential to its unique translesion capability. However, whether the extreme N-terminus of Polκ is required for its activity is unclear. In this work, we constructed two mouse Polκ deletions, which have either a catalytic core (mPolκ1-516) or a core without the first 21-residues (mPolκ22-516), and tested their activities in the replication of normal and BPDE-DNA. These two Polκ deletions are nearly as efficient as the full length protein (Polκ1-852) in normal DNA synthesis. However, steady-state kinetics reveals a significant reduction in efficiency of dCTP incorporation opposite the lesion by Polκ22-516, along with increased frequencies for misinsertion compared with Polκ1-852 The next nucleotide insertion opposite the template C immediately following the BPDE-dG was also examined, and the bypass differences induced by deletions were highlighted in both insertion and extension step. We conclude that the extreme N-terminal part of Polκ is required for the processivity and fidelity of Polκ during translesion synthesis of BPDE-dG lesions.

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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.

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DNA polymerase η (pol η) is best characterized for its ability to perform accurate and efficient translesion DNA synthesis (TLS) through cyclobutane pyrimidine dimers (CPDs). To ensure accurate bypass the polymerase is not only required to select the correct base, but also discriminate between NTPs and dNTPs. Most DNA polymerases have a conserved "steric gate" residue which functions to prevent incorporation of NMPs during DNA synthesis. Here, we demonstrate that the Phe35 residue of Saccharomyces cerevisiae pol η functions as a steric gate to limit the use of ribonucleotides during polymerization both in vitro and in vivo. Unlike the related pol ι enzyme, wild-type pol η does not readily incorporate NMPs in vitro. In contrast, a pol η F35A mutant incorporates NMPs on both damaged and undamaged DNA in vitro with a high degree of base selectivity. An S.cerevisiae strain expressing pol η F35A (rad30-F35A) that is also deficient for nucleotide excision repair (rad1Δ) and the TLS polymerase, pol ζ (rev3Δ), is extremely sensitive to UV-light. The sensitivity is due, in part, to RNase H2 activity, as an isogenic rnh201Δ strain is roughly 50-fold more UV-resistant than its RNH201(+) counterpart. Interestingly the rad1Δ rev3Δ rad30-F35A rnh201Δ strain exhibits a significant increase in the extent of spontaneous mutagenesis with a spectrum dominated by 1bp deletions at runs of template Ts. We hypothesize that the increased mutagenesis is due to rA incorporation at these sites and that the short poly rA tract is subsequently repaired in an error-prone manner by a novel repair pathway that is specifically targeted to polyribonucleotide tracks. These data indicate that under certain conditions, pol η can compete with the cell's replicases and gain access to undamaged genomic DNA. Such observations are consistent with a role for pol η in replicating common fragile sites (CFS) in human cells.

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3-Nitrobenzanthrone (3-NBA), a nitropolyaromatic hydrocarbon (NitroPAH) pollutant in diesel exhaust, is a potent mutagen and carcinogen. After metabolic activation, the primary metabolites of 3-NBA react with DNA to form dG and dA adducts. One of the three major adducts identified is N-(2'-deoxyguanosin-8-yl)-3-aminobenzanthrone (dG(C8-N-ABA)). This bulky adduct likely stalls replicative DNA polymerases but can be traversed by lesion bypass polymerases in vivo. Here, we employed running start assays to show that a site-specifically placed dG(C8-N-ABA) is bypassed in vitro by Sulfolobus solfataricus DNA polymerase IV (Dpo4), a model Y-family DNA polymerase. However, the nucleotide incorporation rate of Dpo4 was significantly reduced opposite both the lesion and the template position immediately downstream from the lesion site, leading to two strong pause sites. To investigate the kinetic effect of dG(C8-N-ABA) on polymerization, we utilized pre-steady-state kinetic methods to determine the kinetic parameters for individual nucleotide incorporations upstream, opposite, and downstream from the dG(C8-N-ABA) lesion. Relative to the replication of the corresponding undamaged DNA template, both nucleotide incorporation efficiency and fidelity of Dpo4 were considerably decreased during dG(C8-N-ABA) lesion bypass and the subsequent extension step. The lower nucleotide incorporation efficiency caused by the lesion is a result of a significantly reduced dNTP incorporation rate constant and modestly weaker dNTP binding affinity. At both pause sites, nucleotide incorporation followed biphasic kinetics with a fast and a slow phase and their rates varied with nucleotide concentration. In contrast, only the fast phase was observed with undamaged DNA. A kinetic mechanism was proposed for the bypass of dG(C8-N-ABA) bypass catalyzed by Dpo4.

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Accurate DNA synthesis in vivo depends on the ability of DNA polymerases to select dNTPs from a nucleotide pool dominated by NTPs. High fidelity replicative polymerases have evolved to efficiently exclude NTPs while copying long stretches of undamaged DNA. However, to bypass DNA damage, cells utilize specialized low fidelity polymerases to perform translesion DNA synthesis (TLS). Of interest is human DNA polymerase ι (pol ι), which has been implicated in TLS of oxidative and UV-induced lesions. Here, we evaluate the ability of pol ι to incorporate NTPs during DNA synthesis. pol ι incorporates and extends NTPs opposite damaged and undamaged template bases in a template-specific manner. The Y39A "steric gate" pol ι mutant is considerably more active in the presence of Mn(2+) compared with Mg(2+) and exhibits a marked increase in NTP incorporation and extension, and surprisingly, it also exhibits increased dNTP base selectivity. Our results indicate that a single residue in pol ι is able to discriminate between NTPs and dNTPs during DNA synthesis. Because wild-type pol ι incorporates NTPs in a template-specific manner, certain DNA sequences may be "at risk" for elevated mutagenesis during pol ι-dependent TLS. Molecular modeling indicates that the constricted active site of wild-type pol ι becomes more spacious in the Y39A variant. Therefore, the Y39A substitution not only permits incorporation of ribonucleotides but also causes the enzyme to favor faithful Watson-Crick base pairing over mutagenic configurations.

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Low fidelity Escherichia coli DNA polymerase V (pol V/UmuD'2C) is best characterized for its ability to perform translesion synthesis (TLS). However, in recA730 lexA(Def) strains, the enzyme is expressed under optimal conditions allowing it to compete with the cell's replicase for access to undamaged chromosomal DNA and leads to a substantial increase in spontaneous mutagenesis. We have recently shown that a Y11A substitution in the "steric gate" residue of UmuC reduces both base and sugar selectivity of pol V, but instead of generating an increased number of spontaneous mutations, strains expressing umuC_Y11A are poorly mutable in vivo. This phenotype is attributed to efficient RNase HII-initiated repair of the misincorporated ribonucleotides that concomitantly removes adjacent misincorporated deoxyribonucleotides. We have utilized the ability of the pol V steric gate mutant to promote incorporation of large numbers of errant ribonucleotides into the E. coli genome to investigate the fundamental mechanisms underlying ribonucleotide excision repair (RER). Here, we demonstrate that RER is normally facilitated by DNA polymerase I (pol I) via classical "nick translation". In vitro, pol I displaces 1-3 nucleotides of the RNA/DNA hybrid and through its 5'→3' (exo/endo) nuclease activity releases ribo- and deoxyribonucleotides from DNA. In vivo, umuC_Y11A-dependent mutagenesis changes significantly in polymerase-deficient, or proofreading-deficient polA strains, indicating a pivotal role for pol I in ribonucleotide excision repair (RER). However, there is also considerable redundancy in the RER pathway in E. coli. Pol I's strand displacement and FLAP-exo/endonuclease activities can be facilitated by alternate enzymes, while the DNA polymerization step can be assumed by high-fidelity pol III. We conclude that RNase HII and pol I normally act to minimize the genomic instability that is generated through errant ribonucleotide incorporation, but that the "nick-translation" activities encoded by the single pol I polypeptide can be undertaken by a variety of back-up enzymes.

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We isolated a pol inhibitor from the cultured mycelia extract of a fungal strain isolated from natural salt from a sea salt pan in Australia, which was identified as 3-O-methylfunicone by spectroscopic analyses. This compound selectively inhibited the activities of mammalian Y-family DNA polymerases (pols) (i.e., pols eta, iota and kappa). Among these pols, human pol kappa activity was most strongly inhibited, with an IC(50) value of 12.5 microM. On the other hand, the compound barely influenced the activities of the other families of mammalian pols, such as A-family (i.e., pol gamma), B-family (i.e., pols alpha, delta and epsilon) or X-family (i.e., pols beta, lambda and terminal deoxynucleotidyl transferase), and showed no effect on the activities of fish pol delta, plant pols, prokaryotic pols and other DNA metabolic enzymes, such as calf primase of pol alpha, human immunodeficiency virus type-1 (HIV-1) reverse transcriptase, human telomerase, T7 RNA polymerase, mouse IMP dehydrogenase (type II), human topoisomerases I and II, T4 polynucleotide kinase or bovine deoxyribonuclease I. This compound also suppressed the growth of two cultured human cancer cell lines, HCT116 (colon carcinoma cells) and HeLa (cervix carcinoma cells), and UV-treated HeLa cells exhibited lower clonogenic survival in the presence of inhibitor.

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