Association between the Herpes Simplex Virus-1 DNA Polymerase and Uracil DNA Glycosylase

Herpes simplex virus-1 (HSV-1) is a large dsDNA virus that encodes its own DNA replication machinery and other enzymes involved in DNA transactions. We recently reported that the HSV-1 DNA polymerase catalytic subunit (UL30) exhibits apurinic/apyrimidinic and 5′-deoxyribose phosphate lyase activities. Moreover, UL30, in conjunction with the viral uracil DNA glycosylase (UL2), cellular apurinic/apyrimidinic endonuclease, and DNA ligase IIIα-XRCC1, performs uracil-initiated base excision repair. Base excision repair is required to maintain genome stability as a means to counter the accumulation of unusual bases and to protect from the loss of DNA bases. Here we show that the HSV-1 UL2 associates with the viral replisome. We identified UL2 as a protein that co-purifies with the DNA polymerase through numerous chromatographic steps, an interaction that was verified by co-immunoprecipitation and direct binding studies. The interaction between UL2 and the DNA polymerase is mediated through the UL30 subunit. Moreover, UL2 co-localizes with UL30 to nuclear viral prereplicative sites. The functional consequence of this interaction is that replication of uracil-containing templates stalls at positions −1 and −2 relative to the template uracil because of the fact that these are converted into non-instructional abasic sites. These findings support the existence of a viral repair complex that may be capable of replication-coupled base excision repair and further highlight the role of DNA repair in the maintenance of the HSV-1 genome.     

Base Excision Repair of DNA: Glycosylases

The review considers the role of base excision repair in maintaining the constancy of genetic information in the cell. The genetic control and biochemical mechanism are described for the first stage of base excision repair, which is catalyzed by specific enzymes, DNA glycosylases.__________Translated from Genetika, Vol. 41, No. 6, 2005, pp. 725–735.Original Russian Text Copyright © 2005 by Korolev.     

Arabidopsis Uracil DNA Glycosylase (UNG) Is Required for Base Excision Repair of Uracil and Increases Plant Sensitivity to 5-Fluorouracil

Uracil in DNA arises by misincorporation of dUMP during replication and by hydrolytic deamination of cytosine. This common lesion is actively removed through a base excision repair (BER) pathway initiated by a uracil DNA glycosylase (UDG) activity that excises the damage as a free base. UDGs are classified into different families differentially distributed across eubacteria, archaea, yeast, and animals, but remain to be unambiguously identified in plants. We report here the molecular characterization of AtUNG (Arabidopsis thaliana uracil DNA glycosylase), a plant member of the Family-1 of UDGs typified by Escherichia coli Ung. AtUNG exhibits the narrow substrate specificity and single-stranded DNA preference that are characteristic of Ung homologues. Cell extracts from atung^−/− mutants are devoid of UDG activity, and lack the capacity to initiate BER on uracil residues. AtUNG-deficient plants do not display any apparent phenotype, but show increased resistance to 5-fluorouracil (5-FU), a cytostatic drug that favors dUMP misincorporation into DNA. The resistance of atung^−/− mutants to 5-FU is accompanied by the accumulation of uracil residues in DNA. These results suggest that AtUNG excises uracil in vivo but generates toxic AP sites when processing abundant U:A pairs in dTTP-depleted cells. Altogether, our findings point to AtUNG as the major UDG activity in Arabidopsis.     

Base Excision Repair

Base excision repair (BER) corrects DNA damage from oxidation, deamination and alkylation. Such base lesions cause little distortion to the DNA helix structure. BER is initiated by a DNA glycosylase that recognizes and removes the damaged base, leaving an abasic site that is further processed by short-patch repair or long-patch repair that largely uses different proteins to complete BER. At least 11 distinct mammalian DNA glycosylases are known, each recognizing a few related lesions, frequently with some overlap in specificities. Impressively, the damaged bases are rapidly identified in a vast excess of normal bases, without a supply of energy. BER protects against cancer, aging, and neurodegeneration and takes place both in nuclei and mitochondria. More recently, an important role of uracil-DNA glycosylase UNG2 in adaptive immunity was revealed. Furthermore, other DNA glycosylases may have important roles in epigenetics, thus expanding the repertoire of BER proteins.Base excision repair (BER) corrects small base lesions that do not significantly distort the DNA helix structure. Such damage typically results from deamination, oxidation, or methylation (Fig. 1). Much of the damage is the result of spontaneous decay of DNA (Lindahl 1993), although similar damage may also be caused by environmental chemicals, radiation, or treatment with cytostatic drugs. BER takes place in nuclei, as well as in mitochondria, largely using different isoforms of proteins or genetically distant proteins. The identification of Escherichia coli uracil-DNA glycosylase (Ung) in 1974 by Tomas Lindahl marks the discovery of BER. Lindahl searched for an enzyme activity that would act on genomic uracil resulting from cytosine deamination. Such an activity was found, but rather unexpectedly, it was not a nuclease. Instead, Lindahl identified an enzyme that cleaved the bond between uracil and deoxyribose. The resulting abasic site (AP-site) was suggested to be further processed by an AP-endonuclease, an exonuclease, a DNA polymerase, and a ligase. Thus, the fundamental steps in the BER pathway were outlined already in the very first paper (Lindahl 1974). Enzymes that cleave the bond between deoxyribose and a modified or mismatched DNA base are now called DNA glycosylases. Collectively these enzymes initiate base excision repair of a large number of base lesions, each recognized by one or a few DNA glycosylases with overlapping specificities.Open in a separate windowFigure 1.Chemistry of common base lesions and abasic sites.This relatively brief review focuses on recent advances in the mechanism and function of BER with a focus on mammalian proteins. The current view is that BER is important in relation to cancer, neurodegeneration, and aging (Jeppesen et al. 2011; Wallace et al. 2012). Because of limited space, we have referred to reviews for the majority of results published more than 6–7 years ago. Also, for more detailed analyses of different aspects of BER, the reader is referred to excellent reviews on BER proteins and pathways published in Huffman et al. (2005), Beard and Wilson (2006), Berti and McCann (2006), Cortázar et al. (2007), Kavli et al. (2007), Sousa et al. (2007), Tubbs et al. (2007), Berger et al. (2008), Robertson et al. (2009), Friedman and Stivers (2010), Wilson et al. (2010), Svilar et al. (2011), and Jacobs and Schar (2012).     

Initial stages of DNA Base Excision Repair in Nucleosomes

Molecular Biology - In mammalian cells, base excision repair (BER) is the main pathway responsible for the correction of a variety of chemically modified DNA bases. DNA packaging in chromatin...     

Interactome of Base and Nucleotide Excision DNA Repair Systems

Molecular Biology - The base and nucleotide excision DNA repair (BER and NER) systems are aimed at removing specific types of damaged DNA, i.e., oxidized, alkylated, or deaminated bases in the case...     

Tin Oxide Nanowires Suppress Herpes Simplex Virus-1 Entry and Cell-to-Cell Membrane Fusion

The advent of nanotechnology has ushered in the use of modified nanoparticles as potential antiviral agents against diseases such as herpes simplex virus 1 and 2 (HSV-1) (HSV-2), human immunodeficiency virus (HIV), monkeypox virus, and hepatitis B virus. Here we describe the application of tin oxide (SnO₂) nanowires as an effective treatment against HSV-1 infection. SnO₂ nanowires work as a carrier of negatively charged structures that compete with HSV-1 attachment to cell bound heparan sulfate (HS), therefore inhibiting entry and subsequent cell-to-cell spread. This promising new approach can be developed into a novel form of broad-spectrum antiviral therapy especially since HS has been shown to serve as a cellular co-receptor for a number of other viruses as well, including the respiratory syncytial virus, adeno-associated virus type 2, and human papilloma virus.     

Base Excision Repair: AP Endonucleases and DNA Polymerases

The DNA base lesions in living cells occur permanently and with high frequency as a result of the action of exogenous and endogenous factors. The main mechanism providing removal of such lesions is base excision repair.     

A Neuron-Specific Host MicroRNA Targets Herpes Simplex Virus-1 ICP0 Expression and Promotes Latency

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A New DNA Break Repair Pathway Involving PARP3 and Base Excision Repair Proteins

Poly(ADP-ribosyl)ation, which is catalyzed by PARP family proteins, is one of the main reactions in the cell response to genomic DNA damage. Massive impact of DNA-damaging agents (such as oxidative stress and ionizing radiation) causes numerous breaks in DNA. In this case, the development of a fast cell response, which allows the genomic DNA integrity to be retained, may be more important than the repair by more accurate but long-term restoration of the DNA structure. This is the first study to show the possibility of eliminating DNA breaks through their PARP3-dependent mono(ADP-ribosyl)ation followed by ligation and repair of the formed ribo-AP sites by the base excision repair (BER) enzyme complex. Taken together, the results of the studies on ADP-ribosylation of DNA and the data obtained in this study suggest that PARP3 may be a component of the DNA break repair system involving the BER enzyme complex.     

Alcohol-induced One-carbon Metabolism Impairment Promotes Dysfunction of DNA Base Excision Repair in Adult Brain

The brain is one of the major targets of chronic alcohol abuse. Yet the fundamental mechanisms underlying alcohol-mediated brain damage remain unclear. The products of alcohol metabolism cause DNA damage, which in conditions of DNA repair dysfunction leads to genomic instability and neural death. We propose that one-carbon metabolism (OCM) impairment associated with long term chronic ethanol intake is a key factor in ethanol-induced neurotoxicity, because OCM provides cells with DNA precursors for DNA repair and methyl groups for DNA methylation, both critical for genomic stability. Using histological (immunohistochemistry and stereological counting) and biochemical assays, we show that 3-week chronic exposure of adult mice to 5% ethanol (Lieber-Decarli diet) results in increased DNA damage, reduced DNA repair, and neuronal death in the brain. These were concomitant with compromised OCM, as evidenced by elevated homocysteine, a marker of OCM dysfunction. We conclude that OCM dysfunction plays a causal role in alcohol-induced genomic instability in the brain because OCM status determines the alcohol effect on DNA damage/repair and genomic stability. Short ethanol exposure, which did not disturb OCM, also did not affect the response to DNA damage, whereas additional OCM disturbance induced by deficiency in a key OCM enzyme, methylenetetrahydrofolate reductase (MTHFR) in Mthfr^+/− mice, exaggerated the ethanol effect on DNA repair. Thus, the impact of long term ethanol exposure on DNA repair and genomic stability in the brain results from OCM dysfunction, and MTHFR mutations such as Mthfr 677C→T, common in human population, may exaggerate the adverse effects of ethanol on the brain.     

Chlamydial Pre-Infection Protects from Subsequent Herpes Simplex Virus-2 Challenge in a Murine Vaginal Super-Infection Model

Chlamydia trachomatis and Herpes Simplex Virus-2 (HSV-2) genital tract co-infections have been reported in humans and studied in vitro but the clinical consequences are unknown. Limited epidemiologic evidence suggests that these co-infections could be more severe than single infections of either pathogen, but the host-pathogen interactions during co-infection remain uncharacterized. To determine whether disease progression and/or pathogen shedding differs between singly-infected and super-infected animals, we developed an in vivo super-infection model in which female BALB/c mice were vaginally infected with Chlamydia muridarum (Cm) followed later by HSV-2. Pre-infection with Chlamydia 3 or 9 days prior to HSV-2 super-infection conferred significant protection from HSV-2-induced neurologic disease and significantly reduced viral recovery compared to HSV-2 singly-infected controls. Neither protection from mortality nor reduced viral recovery were observed when mice were i) super-infected with HSV-2 on day 27 post Cm; ii) infected with UV-irradiated Cm and super-infected with HSV-2; or iii) azithromycin-treated prior to HSV-2 super-infection. Therefore, protection from HSV-2-induced disease requires active infection with viable chlamydiae and is not observed after chlamydial shedding ceases, either naturally or due to antibiotic treatment. Thus, Chlamydia-induced protection is transient and requires the continued presence of chlamydiae or their components. These data demonstrate that chlamydial pre-infection can alter progression of subsequent HSV-2 infection, with implications for HSV-2 transmission from co-infected humans.     

Evidence That Msh1p Plays Multiple Roles in Mitochondrial Base Excision Repair

Mitochondrial DNA is thought to be especially prone to oxidative damage by reactive oxygen species generated through electron transport during cellular respiration. This damage is mitigated primarily by the base excision repair (BER) pathway, one of the few DNA repair pathways with confirmed activity on mitochondrial DNA. Through genetic epistasis analysis of the yeast Saccharomyces cerevisiae, we examined the genetic interaction between each of the BER proteins previously shown to localize to the mitochondria. In addition, we describe a series of genetic interactions between BER components and the MutS homolog MSH1, a respiration-essential gene. We show that, in addition to their variable effects on mitochondrial function, mutant msh1 alleles conferring partial function interact genetically at different points in mitochondrial BER. In addition to this separation of function, we also found that the role of Msh1p in BER is unlikely to be involved in the avoidance of large-scale deletions and rearrangements.DEPLETION of mitochondrial function has been implicated in the human aging process as well as in several inherited and aging-related disorders (Wallace 2005; Weissman et al. 2007). Much of this dysfunction may be attributed to mitochondrial genome instability, as the respiratory capacity of the mitochondria is dependent on an intact genome. Since respiration is essential for the survival of eukaryotic obligate aerobes, the facultative anaerobe Saccharomyces cerevisiae is an ideal model system for mitochondrial studies. Despite the difference in size between the mitochondrial genomes of yeast and humans, the encoded components are required for the same process, cellular energy production (Foury et al. 1998). Therefore, studying how S. cerevisiae maintain mitochondrial DNA (mtDNA) could lend valuable insight into mitochondrial genome maintenance in higher eukaryotes (Perocchi et al. 2008).The necessary process of electron transport during respiration can cause damage to proteins, lipids, and nucleic acids through the formation of reactive oxygen species (ROS) (Longo et al. 1996). Because mtDNA exists in this harsh environment, it is thought that it is especially prone to oxidative damage (Bohr 2002). Damaged bases can be mutagenic by misincorporation opposite the damage by the replicative polymerase or by translesion synthesis beyond the damaged base. Therefore, the repair of oxidative lesions is essential for the stability of the mitochondrial genome.An important mechanism for repair of oxidative DNA damage is the base excision repair (BER) pathway (Croteau and Bohr 1997; Nilsen and Krokan 2001; Bohr 2002). This pathway is well studied in the nucleus of many organisms, and isoforms of several key components have been shown to localize to the mitochondrial compartment (Rosenquist et al. 1997; You et al. 1999; Vongsamphanh et al. 2001). However, despite their extensive nuclear and biochemical characterization, the role of these isoforms in the repair of mtDNA is poorly understood.BER is initiated when an N-glycosylase recognizes a damaged base and cleaves the glycosidic bond between it and the sugar-phosphate backbone, creating an apurinic/apyrimidinic (AP) site that can be repaired by one of two BER pathways. In short patch BER, the AP site is processed by an AP endonuclease on the 5′ side of the damaged base and by the AP lyase activity of a glycosylase, or polymerase β, on the 3′ side of the damage, to create a single-strand gap (Wilson et al. 1998). This gap is filled by a DNA polymerase and then ligated to complete the repair. In the alternative method of long-patch BER, the DNA is again cleaved by an AP endonuclease to generate an available 3′-end for synthesis by a DNA polymerase at the nick, displacing the existing sequence containing the abasic site and creating a 5′ flap. This flap is cleaved by a flap endonuclease, and the resulting nick is sealed by DNA ligase, completing the repair. Biochemical studies suggest that both short-patch and long-patch pathways are active in mitochondria (Akbari et al. 2008; Liu et al. 2008; Szczesny et al. 2008).In this study, we examine the mitochondrial roles of Apn1p, Ntg1p, and Ogg1p, three well-studied BER components. The N-glycosylase Ogg1p is important for the repair of oxidatively damaged DNA, and studies of ogg1-Δ strains have found an increase in point mutations in both nuclear and mitochondrial DNA (Thomas et al. 1997; Singh et al. 2001). In yeast, it was previously demonstrated that a deletion of the N-glycosylase NTG1, or the AP endonuclease APN1, leads to a decrease in mitochondrial mutations as measured by rates of erythromycin resistance, suggesting that the actions of Ntg1p and Apn1p create mutagenic intermediates in mtDNA during repair (Phadnis et al. 2006). This stands in contrast to the increases seen for nuclear DNA mutation rates in the presence of these deletion alleles, indicating that it is not always possible to extrapolate the mitochondrial function of BER proteins on the basis of their nuclear functions, thus making mitochondrial-specific studies necessary (Ramotar et al. 1991, 1993; Alseth et al. 1999; Bennett 1999). In addition, there are likely to be mitochondrial-specific players in the pathway. Here we show that the mismatch repair homolog Msh1p plays multiple roles in mitochondrial BER.Msh1p is the only one of six yeast homologs of MutS, the bacterial mismatch repair protein, which has been found localized to the mitochondria (Reenan and Kolodner 1992; Chi and Kolodner 1994). Msh1p is essential for mitochondrial function and maintenance of mtDNA, necessitating the use of partial function mutants to study the role of Msh1p in mtDNA maintenance (Mookerjee et al. 2005). Although the effects of its disruption have been examined in multiple studies, the mechanism by which Msh1p acts to carry out its essential functions remains unclear (Reenan and Kolodner 1992; Koprowski et al. 2002; Mookerjee et al. 2005; Mookerjee and Sia 2006). Its role as a mitochondrial mismatch repair protein has been disputed, particularly since there are no other mismatch repair proteins that localize to the mitochondria. However, since mtDNA has such a high potential requirement for BER, it is possible that this pathway in the mitochondria may utilize Msh1p. Previous studies have shown genetic interactions between Msh1p and the BER proteins Ogg1p, Apn1p, and Ntg1p (Dzierzbicki et al. 2004; Kaniak et al. 2009). Using msh1 alleles disrupted in conserved DNA binding and ATPase domains, we have examined the frequency and spectrum of mutations responsible for the different mutation rates seen with each allele.     

Impact of Single Nucleotide Polymorphisms of Base Excision Repair Genes on DNA Damage and Efficiency of DNA Repair in Recurrent Depression Disorder

Elevated level of DNA damage was observed in patients with depression. Furthermore, single nucleotide polymorphisms (SNPs) of base excision repair (BER) genes may modulate the risk of this disease. Therefore, the aim of this study was to delineate the association between DNA damage, DNA repair, the presence of polymorphic variants of BER genes, and occurrence of depression. The study was conducted on peripheral blood mononuclear cells of 43 patients diagnosed with depression and 59 controls without mental disorders. Comet assay was used to assess endogenous (oxidative) DNA damage and efficiency of DNA damage repair (DRE). TaqMan probes were employed to genotype 12 SNPs of BER genes. Endogenous DNA damage was higher in the patients than in the controls, but none of the SNPs affected its levels. DRE was significantly higher in the controls and was modulated by BER SNPs, particularly by c.977C>G–hOGG1, c.972G>C–MUTYH, c.2285T>C–PARP1, c.580C>T–XRCC1, c.1196A>G–XRCC1, c.444T>G–APEX1, c.-468T>G–APEX1, or c.*50C>T–LIG3. Our study suggests that both oxidative stress and disorders in DNA damage repair mechanisms contribute to elevated levels of DNA lesions observed in depression. Lower DRE can be partly attributed to the presence of specific SNP variants.