The structure and duplex context of DNA interstrand crosslinks affects the activity of DNA polymerase η.

Several important anti-tumor agents form DNA interstrand crosslinks (ICLs), but their clinical efficiency is counteracted by multiple complex DNA repair pathways. All of these pathways require unhooking of the ICL from one strand of a DNA duplex by nucleases, followed by bypass of the unhooked ICL by translesion synthesis (TLS) polymerases. The structures of the unhooked ICLs remain unknown, yet the position of incisions and processing of the unhooked ICLs significantly influence the efficiency and fidelity of bypass by TLS polymerases. We have synthesized a panel of model unhooked nitrogen mustard ICLs to systematically investigate how the state of an unhooked ICL affects pol η activity. We find that duplex distortion induced by a crosslink plays a crucial role in translesion synthesis, and length of the duplex surrounding an unhooked ICL critically affects polymerase efficiency. We report the synthesis of a putative ICL repair intermediate that mimics the complete processing of an unhooked ICL to a single crosslinked nucleotide, and find that it provides only a minimal obstacle for DNA polymerases. Our results raise the possibility that, depending on the structure and extent of processing of an ICL, its bypass may not absolutely require TLS polymerases.

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.

A strategy for the expression of recombinant proteins traditionally hard to purify.

We have developed a series of plasmid vectors for the soluble expression and subsequent purification of recombinant proteins that have historically proven to be extremely difficult to purify from Escherichia coli. Instead of dramatically overproducing the target protein, it is expressed at a low basal level that facilitates the correct folding of the recombinant protein and increases its solubility. Highly active recombinant proteins that are traditionally difficult to purify are readily purified using standard affinity tags and conventional chromatography. To demonstrate the utility of these vectors, we have expressed and purified full-length human DNA polymerases η, ι, and ν from E. coli and show that the purified DNA polymerases are catalytically active in vitro.

129-derived strains of mice are deficient in DNA polymerase iota and have normal immunoglobulin hypermutation.

Recent studies suggest that DNA polymerase eta (poleta) and DNA polymerase iota (poliota) are involved in somatic hypermutation of immunoglobulin variable genes. To test the role of poliota in generating mutations in an animal model, we first characterized the biochemical properties of murine poliota. Like its human counterpart, murine poliota 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 iota and eta. 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 poliota. 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 poliota-deficient mice. Thus, either poliota does not participate in hypermutation, or its role is nonessential and can be readily assumed by another low-fidelity polymerase.

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 S(N)2 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 alpha and delta. In contrast, human polymerases eta, iota and kappa, as well as Saccharomyces cerevisiae poleta 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, poleta (Rad30) contributes to the survival of cells exposed to methyl methanesulfonate (MMS) and in the absence of Mag1, Rad30 and Rev3, human polymerases eta, iota and kappa are capable of restoring MMS-resistance to the normally MMS-sensitive strain.

Mouse DNA polymerase ι lacking the forty-two amino acids encoded by exon-2 is catalytically inactive in vitro.

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.

DNA polymerase ι: The long and the short of it!

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.

Translesion replication of benzo[a]pyrene and benzo[c]phenanthrene diol epoxide adducts of deoxyadenosine and deoxyguanosine by human DNA polymerase iota.

Human DNA polymerase iota (poliota) is a Y-family polymerase whose cellular function is presently unknown. Here, we report on the ability of poliota to bypass various stereoisomers of benzo[a]pyrene (BaP) diol epoxide (DE) and benzo[c]phenanthrene (BcPh) DE adducts at deoxyadenosine (dA) or deoxyguanosine (dG) bases in four different template sequence contexts in vitro. We find that the BaP DE dG adducts pose a strong block to poliota-dependent replication and result in a high frequency of base misincorporations. In contrast, misincorporations opposite BaP DE and BcPh DE dA adducts generally occurred with a frequency ranging between 2 x 10(-3) and 6 x 10(-4). Although dTMP was inserted efficiently opposite all dA adducts, further extension was relatively poor, with one exception (a cis opened adduct derived from BcPh DE) where up to 58% extension past the lesion was observed. Interestingly, another human Y-family polymerase, polkappa, was able to extend dTMP inserted opposite a BaP DE dA adduct. We suggest that poliota might therefore participate in the error-free bypass of DE-adducted dA in vivo by predominantly incorporating dTMP opposite the damaged base. In many cases, elongation would, however, require the participation of another polymerase more specialized in extension, such as polkappa.

DNA polymerase ι functions in the generation of tandem mutations during somatic hypermutation of antibody genes.

DNA polymerase ι (Pol ι) is an attractive candidate for somatic hypermutation in antibody genes because of its low fidelity. To identify a role for Pol ι, we analyzed mutations in two strains of mice with deficiencies in the enzyme: 129 mice with negligible expression of truncated Pol ι, and knock-in mice that express full-length Pol ι that is catalytically inactive. Both strains had normal frequencies and spectra of mutations in the variable region, indicating that loss of Pol ι did not change overall mutagenesis. We next examined if Pol ι affected tandem mutations generated by another error-prone polymerase, Pol ζ. The frequency of contiguous mutations was analyzed using a novel computational model to determine if they occur during a single DNA transaction or during two independent events. Analyses of 2,000 mutations from both strains indicated that Pol ι-compromised mice lost the tandem signature, whereas C57BL/6 mice accumulated significant amounts of double mutations. The results support a model where Pol ι occasionally accesses the replication fork to generate a first mutation, and Pol ζ extends the mismatch with a second mutation.

Sequence context-dependent replication of DNA templates containing UV-induced lesions by human DNA polymerase iota.

Humans possess four Y-family polymerases: pols eta, iota, kappa and the Rev1 protein. The pivotal role that pol eta plays in protecting us from UV-induced skin cancers is unquestioned given that mutations in the POLH gene (encoding pol eta), lead to the sunlight-sensitive and cancer-prone xeroderma pigmentosum variant phenotype. The roles that pols iota, kappa and Rev1 play in the tolerance of UV-induced DNA damage is, however, much less clear. For example, in vitro studies in which the ability of pol iota to bypass UV-induced cyclobutane pyrimidine dimers (CPDs) or 6-4 pyrimidine-pyrimidone (6-4PP) lesions has been assayed, are somewhat varied with results ranging from limited misinsertion opposite CPDs to complete lesion bypass. We have tested the hypothesis that such discrepancies might have arisen from different assay conditions and local sequence contexts surrounding each UV-photoproduct and find that pol iota can facilitate significant levels of unassisted highly error-prone bypass of a T-T CPD, particularly when the lesion is located in a 3'-A[T-T]A-5' template sequence context and the reaction buffer contains no KCl. When encountering a T-T 6-4PP dimer under the same assay conditions, pol iota efficiently and accurately inserts the correct base, A, opposite the 3'T of the 6-4PP by factors of approximately 10(2) over the incorporation of incorrect nucleotides, while incorporation opposite the 5'T is highly mutagenic. Pol kappa has been proposed to function in the bypass of UV-induced lesions by helping extend primers terminated opposite CPDs. However, we find no evidence that the combined actions of pol iota and pol kappa result in a significant increase in bypass of T-T CPDs when compared to pol iota alone. Our data suggest that under certain conditions and sequence contexts, pol iota can bypass T-T CPDs unassisted and can efficiently incorporate one or more bases opposite a T-T 6-4PP. Such biochemical activities may, therefore, be of biological significance especially in XP-V cells lacking the primary T-T CPD bypassing enzyme, pol eta.

poliota-dependent lesion bypass in vitro.

Based upon phylogenetic relationships, the broad Y-family of DNA polymerases can be divided into various subfamilies consisting of UmuC (polV)-like; DinB (polIV/polkappa)-like; Rev1-like, Rad30A (poleta)-like and Rad30B (poliota)-like polymerases. The polIV/polkappa-like polymerases are most ubiquitous, having been identified in bacteria, archaea and eukaryotes. In contrast, the polV-like polymerases appear restricted to bacteria (both Gram positive and Gram negative). Rev1 and poleta-like polymerases are found exclusively in eukaryotes, and to date, poliota-like polymerases have only been identified in higher eukaryotes. In general, the in vitro properties of polymerases characterized within each sub-family are quite similar. An exception to this rule occurs with the poliota-like polymerases, where the enzymatic properties of Drosophila melanogaster poliota are more similar to that of Saccharomyces cerevisiae and human poleta than to the related human poliota. For example, like poleta, Drosophila poliota can bypass a cis-syn thymine-thymine dimer both accurately and efficiently, while human poliota bypasses the same lesion inefficiently and with low-fidelity. Even in cases where human poliota can efficiently insert a base opposite a lesion (such as a synthetic abasic site, the 3'T of a 6-4-thymine-thymine pyrimidine-pyrimidone photoproduct or opposite benzo[a]pyrene diol epoxide deoxyadenosine adducts), further extension is often limited. Thus, although poliota most likely arose from a genetic duplication of poleta millions of years ago as eukaryotes evolved, it would appear that poliota from humans (and possibly all mammals) has been further subjected to evolutionary pressures that have "tailored" its enzymatic properties away from lesion bypass and towards other function(s) specific for higher eukaryotes. The identification of such functions and the role that mammalian poliota plays in lesion bypass in vivo, should hopefully be forthcoming with the construction of human cell lines deleted for poliota and the identification of mice deficient in poliota.

Increased catalytic activity and altered fidelity of human DNA polymerase iota in the presence of manganese.

All DNA polymerases require a divalent cation for catalytic activity. It is generally assumed that Mg(2+) is the physiological cofactor for replicative DNA polymerases in vivo. However, recent studies suggest that certain repair polymerases, such as pol lambda, may preferentially utilize Mn(2+) in vitro. Here we report on the effects of Mn(2+) and Mg(2+) on the enzymatic properties of human DNA polymerase iota (pol iota). pol iota exhibited the greatest activity in the presence of low levels of Mn(2+) (0.05-0.25 mm). Peak activity in the presence of Mg(2+) was observed in the range of 0.1-0.5 mm and was significantly reduced at concentrations >2 mm. Steady-state kinetic analyses revealed that Mn(2+) increases the catalytic activity of pol iota by approximately 30-60,000-fold through a dramatic decrease in the K(m) value for nucleotide incorporation. Interestingly, whereas pol iota preferentially misinserts G opposite T by a factor of approximately 1.4-2.5-fold over the correct base A in the presence of 0.25 and 5 mm Mg(2+), respectively, the correct insertion of A is actually favored 2-fold over the misincorporation of G in the presence of 0.075 mm Mn(2+). Low levels of Mn(2+) also dramatically increased the ability of pol iota to traverse a variety of DNA lesions in vitro. Titration experiments revealed a strong preference of pol iota for Mn(2+) even when Mg(2+) is present in a >10-fold excess. Our observations therefore raise the intriguing possibility that the cation utilized by pol iota in vivo may actually be Mn(2+) rather than Mg(2+), as tacitly assumed.

Mammalian translesion DNA synthesis across an acrolein-derived deoxyguanosine adduct. Participation of DNA polymerase eta in error-prone synthesis in human cells.

alpha-OH-PdG, an acrolein-derived deoxyguanosine adduct, inhibits DNA synthesis and miscodes significantly in human cells. To probe the cellular mechanism underlying the error-free and error-prone translesion DNA syntheses, in vitro primer extension experiments using purified DNA polymerases and site-specific alpha-OH-PdG were conducted. The results suggest the involvement of pol eta in the cellular error-prone translesion synthesis. Experiments with xeroderma pigmentosum variant cells, which lack pol eta, confirmed this hypothesis. The in vitro results also suggested the involvement of pol iota and/or REV1 in inserting correct dCMP opposite alpha-OH-PdG during error-free synthesis. However, none of translesion-specialized DNA polymerases catalyzed significant extension from a dC terminus when paired opposite alpha-OH-PdG. Thus, our results indicate the following. (i) Multiple DNA polymerases are involved in the bypass of alpha-OH-PdG in human cells. (ii) The accurate and inaccurate syntheses are catalyzed by different polymerases. (iii) A modification of the current eukaryotic bypass model is necessary to account for the accurate bypass synthesis in human cells.