Polymorphisms in DNA repair and environmental interactions

The repair of damage to DNA is critical to the survival of a cell. However, not all organisms nor all individuals express a similar response to challenges to their genetic material. Numerous polymorphisms in genes involved in DNA repair have been found in individuals with DNA repair-related disease as well as in the general population. Studies of these variants are critical in understanding the response of the cell to DNA damage. In some cases, these changes predispose the carrier to a greatly increased risk of cancer. In other cases, the effects are subtler and depend on interactions between the alleles of several genes, or with environmental factors. Consequently, the health effects of exposure to genotoxic or carcinogenic compounds or agents can depend on the variations in these genes. This review will highlight some of the effects that variants, found in many of the genes involved in human DNA repair pathways, have on the response to damage, and their role in susceptibility of the cell and organism to environmental genotoxins. This review will concentrate on the mismatch repair, nucleotide repair, base excision repair, strand break repair, and direct alkyl repair pathways.     

Emerging models for DNA repair: Dictyostelium discoideum as a model for nonhomologous end-joining

DNA double strand breaks (DSBs) are a particularly cytotoxic variety of DNA lesion that can be repaired by homologous recombination (HR) or nonhomologous end-joining (NHEJ). HR utilises sequences homologous to the damage DNA template to facilitate repair. In contrast, NHEJ does not require homologous sequences for repair but instead functions by directly re-joining DNA ends. These pathways are critical to resolve DSBs generated intentionally during processes such as meiotic and site-specific recombination. However, they are also utilised to resolve potentially pathological DSBs generated by mutagens and errors during DNA replication. The importance of DSB repair is underscored by the findings that defects in these pathways results in chromosome instability that contributes to a variety of disease states including malignancy. The general principles of NHEJ are conserved in eukaryotes. As such, relatively simple model organisms have been instrumental in identifying components of these pathways and providing a mechanistic understanding of repair that has subsequently been applied to vertebrates. However, certain components of the NHEJ pathway are absent or show limited conservation in the most commonly used invertebrate models exploited to study DNA repair. Recently, however, it has become apparent that vertebrate DNA repair pathway components, including those involved in NHEJ, are unusually conserved in the amoeba Dictyostelium discoideum. Traditionally, this genetically tractable organism has been exploited to study the molecular basis of cell type specification, cell motility and chemotaxis. Here we discuss the use of this organism as an additional model to study DNA repair, with specific reference to NHEJ.     

Genetic interactions of DNA repair pathways in the pathogen Neisseria meningitidis

The current increase in the incidence and severity of infectious diseases mandates improved understanding of the basic biology and DNA repair profiles of virulent microbes. In our studies of the major pathogen and model organism Neisseria meningitidis, we constructed a panel of mutants inactivating genes involved in base excision repair, mismatch repair, nucleotide excision repair (NER), translesion synthesis, and recombinational repair pathways. The highest spontaneous mutation frequency among the N. meningitidis single mutants was found in the MutY-deficient strain as opposed to mutS mutants in Escherichia coli, indicating a role for meningococcal MutY in antibiotic resistance development. Recombinational repair was recognized as a major pathway counteracting methyl methanesulfonate-induced alkylation damage in the N. meningitidis. In contrast to what has been shown in other species, meningococcal NER did not contribute significantly to repair of alkylation-induced DNA damage, and meningococcal recombinational repair may thus be one of the main pathways for removal of abasic (apurinic/apyrimidinic) sites and strand breaks in DNA. Conversely, NER was identified as the main meningococcal defense pathway against UV-induced DNA damage. N. meningitidis RecA single mutants exhibited only a moderate decrease in survival after UV exposure as opposed to E. coli recA strains, which are extremely UV sensitive, possibly reflecting the lack of a meningococcal SOS response. In conclusion, distinct differences between N. meningitidis and established DNA repair characteristics in E. coli and other species were identified.     

Chemotaxis and flagellar genes of Chromobacterium violaceum

The availability of the complete genome of the Gram-negative beta-proteobacterium Chromobacterium violaceum has increasingly impacted our understanding of this microorganism. This review focuses on the genomic organization and structural analysis of the deduced proteins of the chemosensory adaptation system of C. violaceum. C. violaceum has multiple homologues of most chemotaxis genes, organized mostly in clusters in the bacterial genome. We found at least 67 genes, distributed in 10 gene clusters, involved in the chemotaxis of C. violaceum. A close examination of the chemoreceptors methyl-accepting chemotaxis proteins (MCPs), and the deduced sequences of the members of the two-component signaling system revealed canonical motifs, described as essential for the function of the deduced proteins. The chemoreceptors found in C. violaceum include the complete repertoire of such genes described in bacteria, designated as tsr, tar, trg, and tap; 41 MCP loci were found in the C. violaceum genome. Also, the C. violaceum genome includes a large repertoire of the proteins of the chemosensory transducer system. Multiple homologues of bacterial chemotaxis genes, including CheA, CheB, CheD, CheR, CheV, CheY, CheZ, and CheW, were found in the C. violaceum genome.     

Regulation of the DNA replication fork: a way to fight genomic instability

DNA replication is a complex mechanism that functions due to the coordinated interplay of many factors. In the last few years, numerous studies have suggested that DNA replication factors are closely implicated in several DNA transaction events that maintain the integrity of the genome. Therefore, DNA replication fork factors have to be considered as part of a general process that aims to protect and replicate the genome in order to allow correct functioning of a cell and its eventual daughter cells. This is illustrated by the numerous factors that have a well-defined function at the DNA replication fork, but also play crucial roles in different DNA repair pathways such as base excision repair, nucleotide excision repair, double-strand break repair, and mismatch repair. Moreover, several of the replisome proteins have also been shown to be essential in sensing and transducing DNA damages through the checkpoint cascade pathways, including the recently characterised alternative clamps and clamp-loaders. In this review we present DNA replication factors that are involved in different DNA transaction and checkpoint regulation pathways, with emphasis on the link between DNA replication and maintenance of genomic stability.     

Sirtuin家族与DNA损伤修复

DNA损伤的发生与积累是造成细胞功能紊乱的根本原因,也是引起衰老与肿瘤等疾病发生的关键事件。为维持机体自身遗传物质的完整性与稳定性,生物体内拥有多种针对不同类型DNA损伤的修复方式。Sirtuin蛋白是一组NAD+依赖的、高度保守的组蛋白去乙酰化酶,可通过去乙酰化作用调节众多底物蛋白质的表达、活性与稳定性。 近来的研究显示,DNA损伤修复途径的多个关键蛋白质是Sirtuin的下游底物。Sirtuin蛋白通过调节同源重组修复、非同源末端修复、核苷酸切除修复等途径中的核心蛋白质参与修复包括双链断裂（double stranded breakes, DSBs）在内的多种DNA损伤类型,从而在维持基因组稳定性、寿命以及细胞能量代谢调节等一系列生物学作用中发挥至关重要的作用。本综述将介绍近年来Sirtuin与DNA损伤修复的研究进展。     

Plant homologue of human excision repair gene ERCC1 points to conservation of DNA repair mechanisms

Nucleotide excision repair (NER), a highly versatile DNA repair mechanism, is capable of removing various types of DNA damage including those induced by UV radiation and chemical mutagens. NER has been well characterized in yeast and mammalian systems but its presence in plants has not been reported. Here it is reported that a plant gene isolated from male germline cells of lily (Lilium longiflorum) shows a striking amino acid sequence similarity to the DNA excision repair proteins human ERCC1 and yeast RAD10. Homologous genes are also shown to be present in a number of taxonomically diverse plant genera tested, suggesting that this gene may have a conserved function in plants. The protein encoded by this gene is able to correct significantly the sensitivity to the cross-linking agent mitomycin C in ERCC1-deficient Chinese hamster ovary (CHO) cells. These findings suggest that the NER mechanism is conserved in yeast, animals and higher plants.     

Effects of bleomycin on growth kinetics and survival of Saccharomyces cerevisiae: a model of repair pathways.

In order to analyze the roles of some repair genes in the processing of bleomycin-induced DNA damage and, especially, the interrelationships among the involved repair pathways, we investigated the potentially lethal effect of bleomycin on radiosensitive mutants of Saccharomyces cerevisiae defective in recombination, excision, and RAD6-dependent DNA repair. Using single, double, and triple rad mutants, we analyzed growth kinetics and survival curves as a function of bleomycin concentration. Our results indicate that genes belonging to the three epistasis groups interact in the repair of bleomycin-induced DNA damage to different degrees depending on the concentration of bleomycin. The most important mechanisms involved are recombination and postreplication repair. The initial action of a potentially inducible excision repair gene could provide intermediate substrates for the RAD6- and RAD52-dependent repair processes. Interaction between RAD6 and RAD52 genes was epistatic for low bleomycin concentrations. RAD3 and RAD52 genes act independently in processing DNA damage induced by high concentrations of bleomycin. The synergistic interaction observed at high concentrations in the triple mutant rad2-6 rad6-1 rad52-1 indicates partial independence of the involved repair pathways, with possible common substrates. On the basis of the present results, we propose a heuristic model of bleomycin-induced DNA damage repair.     

Genomic approaches to DNA repair and mutagenesis

DNA damage is a constant threat to cells, causing cytotoxicity as well as inducing genetic alterations. The steady-state abundance of DNA lesions in a cell is minimized by a variety of DNA repair mechanisms, including DNA strand break repair, mismatch repair, nucleotide excision repair, base excision repair, and ribonucleotide excision repair. The efficiencies and mechanisms by which these pathways remove damage from chromosomes have been primarily characterized by investigating the processing of lesions at defined genomic loci, among bulk genomic DNA, on episomal DNA constructs, or using in vitro substrates. However, the structure of a chromosome is heterogeneous, consisting of heavily protein-bound heterochromatic regions, open regulatory regions, actively transcribed genes, and even areas of transient single stranded DNA. Consequently, DNA repair pathways function in a much more diverse set of chromosomal contexts than can be readily assessed using previous methods. Recent efforts to develop whole genome maps of DNA damage, repair processes, and even mutations promise to greatly expand our understanding of DNA repair and mutagenesis. Here we review the current efforts to utilize whole genome maps of DNA damage and mutation to understand how different chromosomal contexts affect DNA excision repair pathways.     

Genomic instability and cancer

Tumorigenesis can be viewed as an imbalance between the mechanisms of cell-cycle control and mutation rates within the genes. Genomic instability is broadly classified into microsatellite instability (MIN) associated with mutator phenotype, and chromosome instability (CIN) recognized by gross chromosomal abnormalities. Three intracellular mechanisms are involved in DNA damage repair that leads to mutator phenotype. They include the nucleotide excision repair (NER), base excision repair (BER) and mismatch repair (MMR). The CIN pathway is typically associated with the accumulation of mutations in tumor suppressor genes and oncogenes. Defects in DNA MMR and CIN pathways are responsible for a variety of hereditary cancer predisposition syndromes including hereditary non-polyposis colorectal carcinoma (HNPCC), Bloom syndrome, ataxia-telangiectasia, and Fanconi anaemia. While there are many genetic contributors to CIN and MIN, there are also epigenetic factors that have emerged to be equally damaging to cell-cycle control. Hypermethylation of tumor suppressor and DNA MMR gene promoter regions, is an epigenetic mechanism of gene silencing that contributes to tumorigenesis. Telomere shortening has been shown to increase genetic instability and tumor formation in mice, underscoring the importance of telomere length and telomerase activity in maintaining genomic integrity. Mouse models have provided important insights for discovering critical pathways in the progression to cancer, as well as to elucidate cross talk among different pathways. This review examines various molecular mechanisms of genomic instability and their relevance to cancer.     

Contribution of base excision repair, nucleotide excision repair, and DNA recombination to alkylation resistance of the fission yeast Schizosaccharomyces pombe

DNA damage is unavoidable, and organisms across the evolutionary spectrum possess DNA repair pathways that are critical for cell viability and genomic stability. To understand the role of base excision repair (BER) in protecting eukaryotic cells against alkylating agents, we generated Schizosaccharomyces pombe strains mutant for the mag1 3-methyladenine DNA glycosylase gene. We report that S. pombe mag1 mutants have only a slightly increased sensitivity to methylation damage, suggesting that Mag1-initiated BER plays a surprisingly minor role in alkylation resistance in this organism. We go on to show that other DNA repair pathways play a larger role than BER in alkylation resistance. Mutations in genes involved in nucleotide excision repair (rad13) and recombinational repair (rhp51) are much more alkylation sensitive than mag1 mutants. In addition, S. pombe mutant for the flap endonuclease rad2 gene, whose precise function in DNA repair is unclear, were also more alkylation sensitive than mag1 mutants. Further, mag1 and rad13 interact synergistically for alkylation resistance, and mag1 and rhp51 display a surprisingly complex genetic interaction. A model for the role of BER in the generation of alkylation-induced DNA strand breaks in S. pombe is discussed.     

Microbial genomics: tropical treasure?

A Brazilian consortium has unveiled the genomic DNA sequence of the purple-pigmented bacterium Chromobacterium violaceum, a dominant component of the tropical soil microbiota. The sequence provides insight into the abundant potential of this organism for biotechnological and pharmaceutical applications.     

Nuclear translocation contributes to regulation of DNA excision repair activities

DNA mutations are circumvented by dedicated specialized excision repair systems, such as the base excision repair (BER), nucleotide excision repair (NER), and mismatch repair (MMR) pathways. Although the individual repair pathways have distinct roles in suppressing changes in the nuclear DNA, it is evident that proteins from the different DNA repair pathways interact [Y. Wang, D. Cortez, P. Yazdi, N. Neff, S.J. Elledge, J. Qin, BASC, a super complex of BRCA1-associated proteins involved in the recognition and repair of aberrant DNA structures, Genes Dev. 14 (2000) 927–939; M. Christmann, M.T. Tomicic, W.P. Roos, B. Kaina, Mechanisms of human DNA repair: an update, Toxicology 193 (2003) 3–34; N.B. Larsen, M. Rasmussen, L.J. Rasmussen, Nuclear and mitochondrial DNA repair: similar pathways? Mitochondrion 5 (2005) 89–108]. Protein interactions are not only important for function, but also for regulation of nuclear import that is necessary for proper localization of the repair proteins. This review summarizes the current knowledge on nuclear import mechanisms of DNA excision repair proteins and provides a model that categorizes the import by different mechanisms, including classical nuclear import, co-import of proteins, and alternative transport pathways. Most excision repair proteins appear to contain classical NLS sequences directing their nuclear import, however, additional import mechanisms add alternative regulatory levels to protein import, indirectly affecting protein function. Protein co-import appears to be a mechanism employed by the composite repair systems NER and MMR to enhance and regulate nuclear accumulation of repair proteins thereby ensuring faithful DNA repair.     

耐辐射球菌DNA双链断裂修复机制研究新进展

耐辐射球菌对于电离辐射等DNA损伤剂具有极强的抗性,能够将同一个基因组中同时产生的高达100个以上的DNA双链断裂在数十小时内高效而精准地进行修复,是研究DNA双链断裂修复机制的重要模式生物。同源重组、非同源末端连接和单链退火途径作为3个主要的修复途径参与了耐辐射球菌基因组DNA双链断裂的修复过程。此外,一系列新发现的重要蛋白质,如Ppr I、Ddr B等对于耐辐射球菌基因组的修复过程同样至关重要。根据本实验室和国内外在这一研究领域近年来的报道,以不同的修复途径为线索,综述该菌DNA双链断裂修复机制的最新研究成果。     

DNA repair in Mycobacterium tuberculosis revisited

Our understanding of Mycobacterium tuberculosis DNA repair mechanisms is still poor compared with that of other bacterial organisms. However, the publication of the first complete M. tuberculosis genome sequence 10 years ago boosted the study of DNA repair systems in this organism. A first step in the elucidation of M. tuberculosis DNA repair mechanisms was taken by Mizrahi and Andersen, who identified homologs of genes involved in the reversal or repair of DNA damage in Escherichia coli and related organisms. Genes required for nucleotide excision repair, base excision repair, recombination, and SOS repair and mutagenesis were identified. Notably, no homologs of genes involved in mismatch repair were identified. Novel characteristics of the M. tuberculosis DNA repair machinery have been found over the last decade, such as nonhomologous end joining, the presence of Mpg, ERCC3 and Hlr – proteins previously presumed to be produced exclusively in mammalian cells – and the recently discovered bifunctional dCTP deaminase:dUTPase. The study of these systems is important to develop therapeutic agents that can counteract M. tuberculosis evolutionary changes and to prevent adaptive events resulting in antibiotic resistance. This review summarizes our current understanding of the M. tuberculosis DNA repair system.