John P. McDonald

Misinsertion and bypass of thymine–thymine dimers by human DNA polymerase ι

Human DNA polymerase ι (polι) is a recently discovered enzyme that exhibits extremely low fidelity on undamaged DNA templates. Here, we show that polι is able to facilitate limited translesion replication of a thymine–thymine cyclobutane pyrimidine dimer (CPD). More importantly, however, the bypass event is highly erroneous. Gel kinetic assays reveal that polι misinserts T or G opposite the 3′ T of the CPD ∼1.5 times more frequently than the correct base, A. While polι is unable to extend the T·T mispair significantly, the G·T mispair is extended and the lesion completely bypassed, with the same efficiency as that of the correctly paired A·T base pair. By comparison, polι readily misinserts two bases opposite a 6‐4 thymine–thymine pyrimidine–pyrimidone photoproduct (6‐4PP), but complete lesion bypass is only a fraction of that observed with the CPD. Our data indicate, therefore, that polι possesses the ability to insert nucleotides opposite UV photoproducts as well as to perform unassisted translesion replication that is likely to be highly mutagenic.

Controlling the subcellular localization of DNA polymerases ι and η via interactions with ubiquitin

Y‐family DNA polymerases have spacious active sites that can accommodate a wide variety of geometric distortions. As a consequence, they are considerably more error‐prone than high‐fidelity replicases. It is hardly surprising, therefore, that the in vivo activity of these polymerases is tightly regulated, so as to minimize their inadvertent access to primer‐termini. We report here that one such mechanism employed by human cells relies on a specific and direct interaction between DNA polymerases ι and η with ubiquitin (Ub). Indeed, we show that both polymerases interact noncovalently with free polyUb chains, as well as mono‐ubiquitinated proliferating cell nuclear antigen (Ub‐PCNA). Mutants of polι (P692R) and polη (H654A) were isolated that are defective in their interactions with polyUb and Ub‐PCNA, whilst retaining their ability to interact with unmodified PCNA. Interestingly, the polymerase mutants exhibit significantly lower levels of replication foci in response to DNA damage, thereby highlighting the biological importance of the polymerase–Ub interaction in regulating the access of the TLS polymerases to stalled replication forks in vivo.

129-derived Strains of Mice Are Deficient in DNA Polymerase ι and Have Normal Immunoglobulin Hypermutation

Recent studies suggest that DNA polymerase η (polη) and DNA polymerase ι (polι) are involved in somatic hypermutation of immunoglobulin variable genes. To test the role of polι in generating mutations in an animal model, we first characterized the biochemical properties of murine polι. Like its human counterpart, murine polι is extremely error-prone when catalyzing synthesis on a variety of DNA templates in vitro. Interestingly, when filling in a 1 base-pair gap, DNA synthesis and subsequent strand displacement was greatest in the presence of both pols ι and η. Genomic sequence analysis of Poli led to the serendipitous discovery that 129-derived strains of mice have a nonsense codon mutation in exon 2 that abrogates production of polι. Analysis of hypermutation in variable genes from 129/SvJ (Poli −/−) and C57BL/6J (Poli +/+) mice revealed that the overall frequency and spectrum of mutation were normal in polι-deficient mice. Thus, either polι does not participate in hypermutation, or its role is nonessential and can be readily assumed by another low-fidelity polymerase.

Identification of a DinB/UmuC homolog in the archeon Sulfolobus solfataricus

To date, eight closely related homologs of the Escherichia coli UmuC protein have been identified. All of these homologs appear to play critical roles in damage-inducible mutagenesis in enterobacteriaceae. Recently, a distantly related UmuC-homolog, DinB, has also been identified in E. coli. Using the polymerase chain reaction together with degenerate primers designed against conserved regions found in UmuC-like proteins, we have identified a new member of the UmuC-superfamily in the archeon Sulfolobus solfataricus. This new homolog shows high sequence similarity to DinB and a lower level of similarity to UmuC. As a consequence, we have called this new gene dbh (dinB homolog). Analysis of approximately 2.7 kb DNA encompassing the dbh region revealed several open reading frames (orfs). One, encoding a putative ribokinase, was located immediately upstream of dbh. This orf overlaps the dbh gene by 4 bp suggesting that both proteins might be coordinately expressed. Further upstream of the ribokinase-dbh locus was another orf encoding a potential ATPase homologous to two uncharacterized S. cerevisiae proteins (YD9346.02c and SC38KCXVI_20) and another E. coli DNA repair protein, RuvB. While this is the first report of a UmuC-like homolog in an archeon, we detected additional homologs using protein sequence comparisons in Gram-positive bacteria, cyanobacteria, and among potential human EST products, indicating that UmuC-related proteins comprise a ubiquitous superfamily of proteins probably involved in DNA repair and mutagenesis.

Altered nucleotide misinsertion fidelity associated with polι‐dependent replication at the end of a DNA template

A hallmark of human DNA polymerase ι (polι) is the asymmetric fidelity of replication at template A and T when the enzyme extends primers annealed to a single‐stranded template. Here, we report on the efficiency and accuracy of polι‐dependent replication at a nick, a gap, the very end of a template and from a mispaired primer. Polι cannot initiate synthesis on a nicked DNA substrate, but fills short gaps efficiently. Surprisingly, polιs ability to blunt‐end a 1 bp recessed terminus is dependent upon the template nucleotide encountered and is highly erroneous. At template G, both C and T are inserted with roughly equal efficiency, whilst at template C, C and A are misinserted 8‐ and 3‐fold more often than the correct base, G. Using substrates containing mispaired primer termini, we show that polι can extend all 12 mispairs, but with differing efficiencies. Polι can also extend a tandem mispair, especially when it is located within a short gap. The enzymatic properties of polι appear consistent with that of a somatic hypermutase and suggest that polι may be one of the low‐fidelity DNA polymerases hypothesized to participate in the hypermutation of immunoglobulin variable genes in vivo.

Eukaryotic Y-family polymerases bypass a 3-methyl-2′-deoxyadenosine analog in vitro and methyl methanesulfonate-induced DNA damage in vivo

N3-methyl-adenine (3MeA) is the major cytotoxic lesion formed in DNA by SN2 methylating agents. The lesion presumably blocks progression of cellular replicases because the N3-methyl group hinders interactions between the polymerase and the minor groove of DNA. However, this hypothesis has yet to be rigorously proven, as 3MeA is intrinsically unstable and is converted to an abasic site, which itself is a blocking lesion. To circumvent these problems, we have chemically synthesized a 3-deaza analog of 3MeA (3dMeA) as a stable phosphoramidite and have incorporated the analog into synthetic oligonucleotides that have been used in vitro as templates for DNA replication. As expected, the 3dMeA lesion blocked both human DNA polymerases α and δ. In contrast, human polymerases η, ι and κ, as well as Saccharomyces cerevisiae polη were able to bypass the lesion, albeit with varying efficiencies and accuracy. To confirm the physiological relevance of our findings, we show that in S. cerevisiae lacking Mag1-dependent 3MeA repair, polη (Rad30) contributes to the survival of cells exposed to methyl methanesulfonate (MMS) and in the absence of Mag1, Rad30 and Rev3, human polymerases η, ι and κ are capable of restoring MMS-resistance to the normally MMS-sensitive strain.

Novel thermostable Y-family polymerases: applications for the PCR amplification of damaged or ancient DNAs

For many years, Taq polymerase has served as the stalwart enzyme in the PCR amplification of DNA. However, a major limitation of Taq is its inability to amplify damaged DNA, thereby restricting its usefulness in forensic applications. In contrast, Y-family DNA polymerases, such as Dpo4 from Sulfolobus solfataricus, can traverse a wide variety of DNA lesions. Here, we report the identification and characterization of five novel thermostable Dpo4-like enzymes from Acidianus infernus, Sulfolobus shibatae, Sulfolobus tengchongensis, Stygiolobus azoricus and Sulfurisphaera ohwakuensis, as well as two recombinant chimeras that have enhanced enzymatic properties compared with the naturally occurring polymerases. The Dpo4-like polymerases are moderately processive, can substitute for Taq in PCR and can bypass DNA lesions that normally block Taq. Such properties make the Dpo4-like enzymes ideally suited for the PCR amplification of damaged DNA samples. Indeed, by using a blend of Taq and Dpo4-like enzymes, we obtained a PCR amplicon from ultraviolet-irradiated DNA that was largely unamplifyable with Taq alone. The inclusion of thermostable Dpo4-like polymerases in PCRs, therefore, augments the recovery and analysis of lesion-containing DNA samples, such as those commonly found in forensic or ancient DNA molecular applications.

Removal of misincorporated ribonucleotides from prokaryotic genomes: an unexpected role for nucleotide excision repair.

Stringent steric exclusion mechanisms limit the misincorporation of ribonucleotides by high-fidelity DNA polymerases into genomic DNA. In contrast, low-fidelity Escherichia coli DNA polymerase V (pol V) has relatively poor sugar discrimination and frequently misincorporates ribonucleotides. Substitution of a steric gate tyrosine residue with alanine (umuC_Y11A) reduces sugar selectivity further and allows pol V to readily misincorporate ribonucleotides as easily as deoxynucleotides, whilst leaving its poor base-substitution fidelity essentially unchanged. However, the mutability of cells expressing the steric gate pol V mutant is very low due to efficient repair mechanisms that are triggered by the misincorporated rNMPs. Comparison of the mutation frequency between strains expressing wild-type and mutant pol V therefore allows us to identify pathways specifically directed at ribonucleotide excision repair (RER). We previously demonstrated that rNMPs incorporated by umuC_Y11A are efficiently removed from DNA in a repair pathway initiated by RNase HII. Using the same approach, we show here that mismatch repair and base excision repair play minimal back-up roles in RER in vivo. In contrast, in the absence of functional RNase HII, umuC_Y11A-dependent mutagenesis increases significantly in ΔuvrA, uvrB5 and ΔuvrC strains, suggesting that rNMPs misincorporated into DNA are actively repaired by nucleotide excision repair (NER) in vivo. Participation of NER in RER was confirmed by reconstituting ribonucleotide-dependent NER in vitro. We show that UvrABC nuclease-catalyzed incisions are readily made on DNA templates containing one, two, or five rNMPs and that the reactions are stimulated by the presence of mispaired bases. Similar to NER of DNA lesions, excision of rNMPs proceeds through dual incisions made at the 8th phosphodiester bond 5′ and 4th–5th phosphodiester bonds 3′ of the ribonucleotide. Ribonucleotides misinserted into DNA can therefore be added to the broad list of helix-distorting modifications that are substrates for NER.

A real-time fluorescence method for enzymatic characterization of specialized human DNA polymerases

Specialized DNA polymerases are involved in DNA synthesis during base-excision repair and translesion synthesis across a wide range of chemically modified DNA templates. Notable features of these enzymes include low catalytic efficiency, low processivity and low fidelity. Traditionally, in vitro studies of these enzymes have utilized radiolabeled substrates and gel electrophoretic separation of products. We have developed a simple homogeneous fluorescence-based method to study the enzymology of specialized DNA polymerases in real time. The method is based on fluorescent reporter strand displacement from a tripartite substrate containing a quencher-labeled template strand, an unlabeled primer and a fluorophore-labeled reporter. With this method, we could follow the activity of human DNA polymerases β, η, ι and κ under different reaction conditions, and we investigated incorporation of the aberrant nucleotide, 8-oxodGTP, as well as bypass of an abasic site or 8-oxoG DNA template lesion in different configurations. Lastly, we demonstrate that the method can be used for small molecule inhibitor discovery and characterization in highly miniaturized settings, and we report the first nanomolar inhibitors of Y-family DNA polymerases ι and η. The fluorogenic method presented here should facilitate mechanistic and inhibitor investigations of these polymerases and is also applicable to the study of highly processive replicative polymerases.

DNA polymerase switching: effects on spontaneous mutagenesis in Escherichia coli

Escherichia coli possesses five known DNA polymerases (pols). Pol III holoenzyme is the cells main replicase, while pol I is responsible for the maturation of Okazaki fragments and filling gaps generated during nucleotide excision repair. Pols II, IV and V are significantly upregulated as part of the cells global SOS response to DNA damage and under these conditions, may alter the fidelity of DNA replication by potentially interfering with the ability of pols I and III to complete their cellular functions. To test this hypothesis, we determined the spectrum of rpoB mutations arising in an isogenic set of mutL strains differentially expressing the chromosomally encoded pols. Interestingly, mutagenic hot spots in rpoB were identified that are susceptible to the actions of pols I–V. For example, in a recA730 lexA(Def) mutL background most transversions were dependent upon pols IV and V. In contrast, transitions were largely dependent upon pol I and to a lesser extent, pol III. Furthermore, the extent of pol I‐dependent mutagenesis at one particular site was modulated by pols II and IV. Our observations suggest that there is considerable interplay among all five E. coli polymerases that either reduces or enhances the mutagenic load on the E. coli chromosome.