Biochemical and photochemical mechanisms that produce different UV-induced mutation spectra.

Although UV-induced mutagenesis has been studied extensively, the precise mechanisms that convert UV-induced DNA damage into mutations remain elusive. One well-studied mechanism involves DNA polymerase (Pol) η and ζ, which produces C > T transitions during translesion synthesis (TLS) across pyrimidine dimers. We previously proposed another biochemical mechanism that involves multiple UV-irradiations with incubation in the dark in between. The incubation facilitates spontaneous deamination of cytosine in a pyrimidine dimer, and the subsequent UV irradiation induces photolyase-independent (direct) photoreversal that converts cytosine into monomeric uracil residue. In this paper, we first demonstrate that natural sunlight can induce both mutational processes in vitro. The direct photoreversal was also reproduced by monochromatic UVB at 300 nm. We also demonstrate that post-irradiation incubation in the dark is required for both mutational processes, suggesting that cytosine deamination is required for both the Pol η/ζ-dependent and the photoreversal-dependent mechanisms. Another Y-family polymerase Pol ι also mediated a mutagenic TLS on UV-damaged templates when combined with Pol ζ. The Pol ι-dependent mutations were largely independent of post-irradiation incubation, indicating that cytosine deamination was not essential for this mutational process. Sunlight-exposure also induced C > A transversions which were likely caused by oxidation of guanine residues. Finally, we constructed in vitro mutation spectra in a comparable format to cancer mutation signatures. While both Pol η-dependent and photoreversal-dependent spectra showed high similarities to a cancer signature (SBS7a), Pol ι-dependent mutation spectrum has distinct T > A/C substitutions, which are found in another cancer signature (SBS7d). The Pol ι-dependent T > A/C substitutions were resistant to T4 pyrimidine dimer glycosylase-treatment, suggesting that this mutational process is independent of cis-syn pyrimidine dimers. An updated model about multiple mechanisms of UV-induced mutagenesis is discussed.

Genetic diversity of the human adenovirus species C DNA polymerase.

BACKGROUND: Human Adenovirus (HAdV) are responsible for severe infections in hematopoietic stem cells transplant (HSCT) recipient, species C viruses being the most commonly observed in this population. There is no approved antiviral treatment yet. Cidofovir (CDV), a cytidine analog, is the most frequently used and its lipophilic conjugate, brincidofovir (BCV), is under clinical development. These drugs target the viral DNA polymerase (DNA pol). Little is known about the natural polymorphism of HAdV DNA pol in clinical strains. METHODS: We assessed the inter- and intra-species variability of the whole gene coding for HAdV DNA pol of HAdV clinical strains of species C. The study included 60 species C HAdV (21 C1, 27 C2 and 12 C5) strains isolated from patients with symptomatic infections who had never experienced CDV or BCV treatments and 20 reference strains. We also evaluated the emergence of mutations in thrirteen patients with persistent HAdV infection despite antiviral treatment. RESULTS: We identified 356 polymorphic nucleotide positions (9.9% of the whole gene), including 102 positions with nonsynonymous mutations (28.0%) representing 8.7% of all amino acids. The mean numbers of nucleotide and amino acid mutations per strain were 23.1 (±6.2) and 5.2 (±2.4) respectively. Most of amino acid substitutions (60.6%) were observed in one instance only. A minority (13.8%) were observed in more than 10% of all strains. The most variable region was the NH2 terminal domain (44.2% of amino acid mutations). Mutations in the exonuclease domain accounted for 27.8%. The binding domains for the terminal protein (TPR), TPR1 and TPR2, presented a limited number of mutations, which were nonetheless frequently observed (62.5% and 58.8% of strains for TPR1 and TPR2, respectively). None of the mutations associated with CDV or BCV resistance were detected. In patients receieving antiviral drugs with persistent HAdV replication, we identified a new mutation in the NH2 terminal region. CONCLUSIONS: Our study shows a high diversity in HAdV DNA pol sequences in clinical species C HAdV and provides a comprehensive mapping of its natural polymorphism. These data will contribute to the interpretation of HAdV DNA pol mutations selected in patients receiving antiviral treatments.

Translesion and Repair DNA Polymerases: Diverse Structure and Mechanism.

The number of DNA polymerases identified in each organism has mushroomed in the past two decades. Most newly found DNA polymerases specialize in translesion synthesis and DNA repair instead of replication. Although intrinsic error rates are higher for translesion and repair polymerases than for replicative polymerases, the specialized polymerases increase genome stability and reduce tumorigenesis. Reflecting the numerous types of DNA lesions and variations of broken DNA ends, translesion and repair polymerases differ in structure, mechanism, and function. Here, we review the unique and general features of polymerases specialized in lesion bypass, as well as in gap-filling and end-joining synthesis.

Involvement of translesion synthesis DNA polymerases in DNA interstrand crosslink repair.

DNA interstrand crosslinks (ICLs) covalently join the two strands of a DNA duplex and block essential processes such as DNA replication and transcription. Several important anti-tumor drugs such as cisplatin and nitrogen mustards exert their cytotoxicity by forming ICLs. However, multiple complex pathways repair ICLs and these are thought to contribute to the development of resistance towards ICL-inducing agents. While the understanding of many aspects of ICL repair is still rudimentary, studies in recent years have provided significant insights into the pathways of ICL repair. In this perspective we review the recent advances made in elucidating the mechanisms of ICL repair with a focus on the role of TLS polymerases. We describe the emerging models for how these enzymes contribute to and are regulated in ICL repair, discuss the key open questions and examine the implications for this pathway in anti-cancer therapy.

Biological and therapeutic relevance of nonreplicative DNA polymerases to cancer.

Apart from surgical approaches, the treatment of cancer remains largely underpinned by radiotherapy and pharmacological agents that cause damage to cellular DNA, which ultimately causes cancer cell death. DNA polymerases, which are involved in the repair of cellular DNA damage, are therefore potential targets for inhibitors for improving the efficacy of cancer therapy. They can be divided, according to their main function, into two groups, namely replicative and nonreplicative enzymes. At least 15 different DNA polymerases, including their homologs, have been discovered to date, which vary considerably in processivity and fidelity. Many of the nonreplicative (specialized) DNA polymerases replicate DNA in an error-prone fashion, and they have been shown to participate in multiple DNA damage repair and tolerance pathways, which are often aberrant in cancer cells. Alterations in DNA repair pathways involving DNA polymerases have been linked with cancer survival and with treatment response to radiotherapy or to classes of cytotoxic drugs routinely used for cancer treatment, particularly cisplatin, oxaliplatin, etoposide, and bleomycin. Indeed, there are extensive preclinical data to suggest that DNA polymerase inhibition may prove to be a useful approach for increasing the effectiveness of therapies in patients with cancer. Furthermore, specialized DNA polymerases warrant examination of their potential use as clinical biomarkers to select for particular cancer therapies, to individualize treatment for patients.

Thermodynamic and mechanistic insights into translesion DNA synthesis catalyzed by Y-family DNA polymerase across a bulky double-base lesion of an antitumor platinum drug.

To determine how the Y-family translesion DNA polymerase η (Polη) processes lesions remains fundamental to understanding the molecular origins of the mutagenic translesion bypass. We utilized model systems employing a DNA double-base lesion derived from 1,2-GG intrastrand cross-links of a new antitumor Pt(II) complex containing a bulky carrier ligand, namely [PtCl(2)(cis-1,4-dach)] (DACH=diaminocyclohexane). The catalytic efficiency of Polη for the insertion of correct dCTP, with respect to the other incorrect nucleotides, opposite the 1,2-GG cross-link was markedly reduced by the DACH carrier ligand. This reduced efficiency of Polη to incorporate the correct dCTP could be due to a more extensive DNA unstacking and deformation of the minor groove induced in the DNA by the cross-link of bulky [PtCl(2)(cis-1,4-dach)]. The major products of the bypass of this double-base lesion produced by [PtCl(2)(cis-1,4-dach)] by Polη resulted from misincorporation of dATP opposite the platinated G residues. The results of the present work support the thesis that this misincorporation could be due to sterical effects of the bulkier 1,4-DACH ligand hindering the formation of the Polη-DNA-incoming nucleotide complex. Calorimetric analysis suggested that thermodynamic factors may contribute to the forces that governed enhanced incorporation of the incorrect dATP by Polη as well.

ß-Sitosteryl (6'-O-linoleoyl)-glucoside of soybean (Glycine max L.) crude extract inhibits Y-family DNA polymerases.

In the screening of selective DNA polymerase (pol) inhibitors, we isolated an acylated steryl glycoside, ß-sitosteryl (6'-O-linoleoyl)-glucoside (compound 1), from the waste extract of soybean (Glycine max L.) oil. This compound exhibited a marked ability to inhibit the activities of eukaryotic Y-family pols (pols η, ι and κ), which are repair-related pols. Among mammalian Y-family pols, the activity of mouse pol κ was most strongly inhibited by compound 1, with an IC(50) value of 10.2 µM. On the other hand, compound 1 had no effect on the activities of other eukaryotic pols such as A-family (pol γ), B-family (pols α, δ, and ε), or X-family (pols ß, λ and terminal deoxynucleotidyl transferase) pols. In addition, compound 1 had no effect on prokaryotic pols or other DNA metabolic enzymes such as calf primase of pol α, T7 RNA polymerase, T4 polynucleotide kinase, or bovine deoxyribonuclease I. Compound 1 consists of 3 groups: ß-sitosteryl (compound 2), linoleic acid (compound 3), and D-glucose (compound 4). Compound 3 inhibited the activities of all mammalian pols tested, but compounds 2 and 4 did not have any effect on the tested pols. Kinetic studies showed that the inhibition of pol κ activity by compound 1 was noncompetitive with both the DNA template-primer and nucleotide substrate, whereas compound 3-induced inhibition was competitive with the DNA template-primer and noncompetitive with the nucleotide substrate. The relationship between the structure of compound 1 and the selective inhibition of eukaryotic Y-family pols is discussed.

Y-family DNA polymerases in mammalian cells.

Eukaryotic genomes are replicated with high fidelity to assure the faithful transmission of genetic information from one generation to the next. The accuracy of replication relies heavily on the ability of replicative DNA polymerases to efficiently select correct nucleotides for the polymerization reaction and, using their intrinsic exonuclease activities, to excise mistakenly incorporated nucleotides. Cells also possess a variety of specialized DNA polymerases that, by a process called translesion DNA synthesis (TLS), help overcome replication blocks when unrepaired DNA lesions stall the replication machinery. This review considers the properties of the Y-family (a subset of specialized DNA polymerases) and their roles in modulating spontaneous and genotoxic-induced mutations in mammals. We also review recent insights into the molecular mechanisms that regulate PCNA monoubiquitination and DNA polymerase switching during TLS and discuss the potential of using Y-family DNA polymerases as novel targets for cancer prevention and therapy.

Replication bypass of the acrolein-mediated deoxyguanine DNA-peptide cross-links by DNA polymerases of the DinB family.

DNA-protein cross-links (adducts) are formed in cellular DNA under a variety of conditions, particularly following exposure to an alpha,beta-unsaturated aldehyde, acrolein. DNA-protein cross-links are subject to repair or damage-tolerance processes. These adducts serve as substrates for proteolytic degradation, yielding DNA-peptide lesions that have been shown to be actively repaired by the nucleotide excision repair complex. Alternatively, DNA-peptide cross-links can be subjected to replication bypass. We present new evidence about the capabilities of DNA polymerases to synthesize DNA past such cross-links. DNAs were constructed with site-specific cross-links, in which either a tetrapeptide or a dodecylpeptide was covalently attached at the N (2) position of guanine via an acrolein adduct, and replication bypass assays were carried out with members of the DinB family of polymerases, human polymerase (pol) kappa, Escherichia coli pol IV, and various E. coli polymerases that do not belong to the DinB family. Pol kappa was able to catalyze both the incorporation and the extension steps with an efficiency that was qualitatively indistinguishable from control (undamaged) substrates. Fidelity was comparable on all of these substrates, suggesting that pol kappa would have a role in the low mutation frequency associated with replication of these adducts in mammalian cells. When the E. coli orthologue of pol kappa, damage-inducible DNA polymerase, pol IV, was analyzed on the same substrates, pause sites were detected opposite and three nucleotides beyond the site of the lesion, with incorporation opposite the lesion being accurate. In contrast, neither E. coli replicative polymerase, pol III, nor E. coli damage-inducible polymerases, pol II and pol V, could efficiently incorporate a nucleotide opposite the DNA-peptide cross-links. Consistent with a role for pol IV in tolerance of these lesions, the replication efficiency of DNAs containing DNA-peptide cross-links was greatly reduced in pol IV-deficient cells. Collectively, these data indicate an important role for the DinB family of polymerases in tolerance mechanisms of N (2)-guanine-linked DNA-peptide cross-links.

Additive effects of POLG1 and ANT1 mutations in a complex encephalomyopathy.

MtDNA instability is associated with a wide spectrum of clinical presentations, from dominant or recessive progressive external ophthalmoplegia (PEO) to juvenile-onset spino-cerebellar ataxia and epilepsy (SCAE) or infantile Alpers-Huttenlocher syndrome. We present here the clinical and molecular features of a patient with a clinical presentation characterized initially by PEO with mtDNA multiple deletions lately evolving into a severe neurological syndrome, which included sensory and cerebellar ataxia, peripheral neuropathy, parkinsonism, and depression. This complex phenotype is the result of mutations in two distinct proteins, ANT1 and PolgammaA, which cause additive, deleterious effects on mtDNA maintenance and integrity.

DNA repair by polymerase delta in Saccharomyces cerevisiae is not controlled by the proliferating cell nuclear antigen-like Rad17/Mec3/Ddc1complex

DNA damage activates several mechanisms such as DNA repair and cell cycle checkpoints. The Saccharomyces cerevisiae heterotrimeric checkpoint clamp consisting of the Rad17, Mec3 and Ddc1 subunits is an early response factor to DNA damage and activates checkpoints. This complex is structurally similar to the proliferating cell nuclear antigen (PCNA), which serves as a sliding clamp platform for DNA replication. Growing evidence suggests that PCNA-like complexes play a major role in DNA repair as they have been shown to interact with and stimulate several proteins, including specialized DNA polymerases. With the aim of extending our knowledge concerning the link between checkpoint activation and DNA repair, we tested the possibility of a functional interaction between the Rad17/Mec3/Ddc1 complex and the replicative DNA polymerases alpha, delta and epsilon. The analysis of sensitivity response of single and double mutants to UVC and 8-MOP + UVA-induced DNA damage suggests that the PCNA-like component Mec3p of S. cerevisiae neither relies on nor competes with the third subunit of DNA polymerase delta, Pol32p, for lesion removal. No enhanced sensitivity was observed when inactivating components of DNA polymerases alpha and epsilon in the absence of Mec3p. The hypersensitivity of pol32delta to photoactivated 8-MOP suggests that the replicative DNA polymerase delta also participates in the repair of mono- and bi-functional DNA adducts. Repair of UVC and 8-MOP + UVA-induced DNA damage via polymerase delta thus occurs independent of the Rad17/Mec3/Ddc1 checkpoint clamp.

Werner syndrome protein interacts functionally with translesion DNA polymerases.

Werner syndrome (WS) is characterized by premature onset of age-associated disorders and predisposition to cancer. The WS protein, WRN, encodes 3' --> 5' DNA helicase and 3' --> 5' DNA exonuclease activities, and is implicated in the maintenance of genomic stability. Translesion (TLS) DNA polymerases (Pols) insert nucleotides opposite replication-blocking DNA lesions and presumably prevent replication fork stalling/collapse. Here, we present in vitro and in vivo data that demonstrate functional interaction between WRN and the TLS Pols, Poleta, Polkappa, and Poliota. In vitro, WRN stimulates the extension activity of TLS Pols on lesion-free and lesion-containing DNA templates, and alleviates pausing at stalling lesions. Stimulation is mediated through an increase in the apparent V(max) of the polymerization reaction. Notably, by accelerating the rate of nucleotide incorporation, WRN increases mutagenesis by Poleta. In vivo, WRN and Poleta colocalize at replication-dependent foci in response to UVC irradiation. The functional interaction between WRN and TLS Pols may promote replication fork progression, at the expense of increased mutagenesis, and obviate the need to resolve stalled/collapsed forks by processes involving chromosomal rearrangements.

DNA lesions and repair in immunoglobulin class switch recombination and somatic hypermutation.

Immunoglobulin (Ig) gene somatic hypermutation (SHM) and class switch DNA recombination (CSR) are critical for the maturation of the antibody response. These processes endow antibodies with increased antigen-binding affinity and acquisition of new biological effector functions, thereby underlying the generation of memory B cells and plasma cells. They are dependent on the generation of specific DNA lesions and the intervention of activation-induced cytidine deaminase as well as newly identified translesion DNA polymerases, which are expressed in germinal center B cells. DNA lesions include mismatches, abasic sites, nicks, single-strand breaks, and double-strand breaks (DSBs). DSBs in the switch (S) region DNA are critical for CSR, but they also occur in V(D)J regions and possibly contribute to the events that lead to SHM. The nature of the DSBs in the Ig locus, their generation, and the repair processes that they trigger and that are responsible for their regulation remain poorly understood. Aberrant regulation of these events can result in chromosomal breaks and translocations, which are significant steps in B-cell neoplastic transformation.

Nucleotide sequence and phylogenetic analyses of the DNA polymerase gene of Anticarsia gemmatalis nucleopolyhedrovirus.

The DNA polymerase from Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV) was identified and sequenced, and its amino acid sequence was compared with other viral DNA polymerases to identify conserved regions and to reconstruct a phylogenetic tree. The sequence analysis of the AgMNPV DNA polymerase gene revealed the presence of a 2976 nucleotides open reading frame (ORF) encoding a polypeptide of 991 amino acid residues with a predicted molecular mass of 114.7 kDa. Among the baculovirus DNA polymerase genes identified to date, the AgMNPV DNA polymerase gene shared maximum amino acid sequence identity with the DNA polymerase gene of Choristoneura fumiferana nucleopolyhedrovirus defective strain (CfDEFNPV) (94%). The alignment of 140 virus sequences, 23 of them from baculovirus, showed that, of the 10 conserved regions identified, 5 are exclusive to baculoviruses (R1, R5, R9, R6 and R10), only 2 of them (R6 and R10) previously described as such in the literature. Our analysis, based on their positions in the AgMNPV DNA polymerase model, suggests that R9 and R10 could interact with DNA. Phylogenetic analysis of DNA polymerase sequences places the enzyme from AgMNPV within the cluster containing the polymerases of Group I Nucleopolyhedrovirus and suggests that the AgMNPV DNA polymerase is more closely related to that of CfDEFNPV than to those of other baculoviruses.

Probing minor groove recognition contacts by DNA polymerases and reverse transcriptases using 3-deaza-2'-deoxyadenosine.

Standard nucleobases all present electron density as an unshared pair of electrons to the minor groove of the double helix. Many heterocycles supporting artificial genetic systems lack this electron pair. To determine how different DNA polymerases use the pair as a substrate specificity determinant, three Family A polymerases, three Family B polymerases and three reverse transcriptases were examined for their ability to handle 3-deaza-2'-deoxyadenosine (c3dA), an analog of 2'-deoxyadenosine lacking the minor groove electron pair. Different polymerases differed widely in their interaction with c3dA. Most notably, Family A and Family B polymerases differed in their use of this interaction to exploit their exonuclease activities. Significant differences were also found within polymerase families. This plasticity in polymerase behavior is encouraging to those wishing to develop a synthetic biology based on artificial genetic systems. The differences also suggest either that Family A and Family B polymerases do not share a common ancestor, that minor groove contact was not used by that ancestor functionally or that this contact was not sufficiently critical to fitness to have been conserved as the polymerase families diverged. Each interpretation is significant for understanding the planetary biology of polymerases.

Diversity of structure and function of DNA polymerase (gp43) of T4-related bacteriophages.

The replication DNA polymerase (gp43) of the bacteriophage T4 is a member of the pol B family of DNA polymerases, which are found in all divisions of life in the biosphere. The enzyme is a modularly organized protein that has several activities in one polypeptide chain (approximately 900 amino acid residues). These include two catalytic functions, POL (polymerase) and EXO (3 -exonuclease), and specific binding activities to DNA, the mRNA for gp43, deoxyribonucleotides (dNTPs), and other T4 replication proteins. The gene for this multifunctional enzyme (gene 43) has been preserved in evolution of the diverse group of T4-like phages in nature, but has diverged in sequence, organization, and specificity of the binding functions of the gene product. We describe here examples of T4-like phages where DNA rearrangements have created split forms of gene 43 consisting of two cistrons instead of one. These gene 43 variants specify separate gp43A (N-terminal) and gp43B (C-terminal) subunits of a split form of gp43. Compared to the monocistronic form, the interruption in contiguity of the gene 43 reading frame maps in a highly diverged sequence separating the code for essential components of two major modules of this pol B enzyme, the FINGERS and PALM domains, which contain the dNTP binding pocket and POL catalytic residues of the enzyme. We discuss the biological implications of these gp43 splits and compare them to other types of pol B splits in nature. Our studies suggest that DNA mobile elements may allow genetic information for pol B modules to be exchanged between organisms.

Genetic and ultrastructural characterization of a European isolate of the fatal endotheliotropic elephant herpesvirus.

A male Asian elephant (Elephas maximus) died at the Berlin zoological gardens in August 1998 of systemic infection with the novel endotheliotropic elephant herpesvirus (ElHV-1). This virus causes a fatal haemorrhagic disease in Asian elephants, the so-called endothelial inclusion body disease, as reported from North American zoological gardens. In the present work, ElHV-1 was visualized ultrastructurally in affected organ material. Furthermore, a gene block comprising the complete glycoprotein B (gB) and DNA polymerase (DPOL) genes as well as two partial genes was amplified by PCR-based genome walking and sequenced. The gene content and arrangement were similar to those of members of the Betaherpesvirinae. However, phylogenetic analysis with gB and DPOL consistently revealed a very distant relationship to the betaherpesviruses. Therefore, ElHV-1 may be a member of a new genus or even a new herpesvirus subfamily. The sequence information generated was used to set up a nested-PCR assay for diagnosis of suspected cases of endothelial inclusion body disease. Furthermore, it will aid in the development of antibody-based detection methods and of vaccination strategies against this fatal herpesvirus infection in the endangered Asian elephant.

DNA damage-induced mutagenesis : a novel target for cancer prevention.

Tolerance to some degree of unrepaired DNA damage is crucial for cell survival-more specifically, for the sustained functionality of the DNA replication machinery-in the presence of adverse (genotoxic) conditions. At least two mechanisms ensure such tolerance: template switching and lesion bypass. Lesion bypass, whereby unrepaired damaged DNA serves as template, involves the Y family of DNA polymerases; lesion bypass can be error-free or error-prone, depending on the nucleotide incorporated during translesion synthesis. Error-prone lesion bypass constitutes a major mechanism of mutagenesis and, in eukaryotes, is primarily effected by the DNA polymerase zeta (Polzeta) pathway. A relationship between the Y family polymerases and the Polzeta pathway is thus implicated, and conforms to the two-polymerase two-step model of lesion bypass. Based on the mutagenesis hypothesis of cancer formation, DNA damage-induced mutagenesis and its underlying molecular biology offer an intriguing potential target for cancer prevention.

Two novel human and mouse DNA polymerases of the polX family.

We describe here two novel mouse and human DNA polymerases: one (pol lambda) has homology with DNA polymerase beta while the other one (pol mu) is closer to terminal deoxynucleotidyltransferase. However both have DNA polymerase activity in vitro and share similar structural organization, including a BRCT domain, helix-loop-helix DNA-binding motifs and polymerase X domain. mRNA expression of pol lambda is highest in testis and fetal liver, while expression of pol mu is more lymphoid, with highest expression both in thymus and tonsillar B cells. An unusually large number of splice variants is observed for the pol mu gene, most of which affect the polymerase domain. Expression of mRNA of both polymerases is down-regulated upon treatment by DNA damaging agents (UV light, gamma-rays or H(2)O(2)). This suggests that their biological function may differ from DNA translesion synthesis, for which several DNA polymerase activities have been recently described. Possible functions are discussed.