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.     

The relative roles of three DNA repair pathways in preventing Caenorhabditis elegans mutation accumulation

Mutation is a central biological process whose rates and spectra are influenced by a variety of complex and interacting forces. Although DNA repair pathways are generally known to play key roles in maintaining genetic stability, much remains to be understood about the relative roles of different pathways in preventing the accumulation of mutations and the extent of heterogeneity in pathway-specific repair efficiencies across different genomic regions. In this study we examine mutation processes in base excision repair-deficient (nth-1) and nucleotide excision repair-deficient (xpa-1) Caenorhabditis elegans mutation-accumulation (MA) lines across 24 regions of the genome and compare our observations to previous data from mismatch repair-deficient (msh-2 and msh-6) and wild-type (N2) MA lines. Drastic variation in both average and locus-specific mutation rates, ranging two orders of magnitude for the latter, was detected among the four sets of repair-deficient MA lines. Our work provides critical insights into the relative roles of three DNA repair pathways in preventing C. elegans mutation accumulation and provides evidence for the presence of pathway-specific DNA repair territories in the C. elegans genome.     

Types, levels and patterns of low-copy DNA sequence divergence, and phylogenetic implications, for Gossypium genome types

To explore types, levels and patterns of genetic divergence among diploid Gossypium (cotton) genomes, 780 cDNA, genomic DNA and simple sequence repeat (SSR) loci were re-sequenced in Gossypium herbaceum (A1 genome), G. arboreum (A2), G. raimondii (D5), G. trilobum (D8), G. sturtianum (C1) and an outgroup, Gossypioides kirkii. Divergence among these genomes ranged from 7.32 polymorphic base pairs per 100 between G. kirkii and G. herbaceum (A1) to only 1.44 between G. herbaceum (A1) and G. arboreum (A2). SSR loci are least conserved with 12.71 polymorphic base pairs and 3.77 polymorphic sites per 100 base pairs, whereas expressed sequence tags are most conserved with 3.96 polymorphic base pairs and 2.06 sites. SSR loci also exhibit the highest percentage of 'extended polymorphisms' (spanning multiple consecutive nucleotides). The A genome lineage was particularly rapidly evolving, with the D genome also showing accelerated evolution relative to the C genome. Unexpected asymmetry in mutation rates was found, with much more transition than transversion mutation in the D genome after its divergence from a common ancestor shared with the A genome. This large quantity of orthologous DNA sequence strongly supports a phylogeny in which A-C divergence is more recent than A-D divergence, a subject that is of much importance in view of A-D polyploid formation being key to the evolution of the most productive and finest-quality cottons. Loci that are monomorphic within A or D genome types, but polymorphic between genome types, may be of practical importance for identifying locus-specific DNA markers in tetraploid cottons including leading cultivars.     

Characterization of DNA Repair Deficient Strains of Chlamydomonas reinhardtii Generated by Insertional Mutagenesis

While the mechanisms governing DNA damage response and repair are fundamentally conserved, cross-kingdom comparisons indicate that they differ in many aspects due to differences in life-styles and developmental strategies. In photosynthetic organisms these differences have not been fully explored because gene-discovery approaches are mainly based on homology searches with known DDR/DNA repair proteins. Here we performed a forward genetic screen in the green algae Chlamydomonas reinhardtii to identify genes deficient in DDR/DNA repair. We isolated five insertional mutants that were sensitive to various genotoxic insults and two of them exhibited altered efficiency of transgene integration. To identify genomic regions disrupted in these mutants, we established a novel adaptor-ligation strategy for the efficient recovery of the insertion flanking sites. Four mutants harbored deletions that involved known DNA repair factors, DNA Pol zeta, DNA Pol theta, SAE2/COM1, and two neighbouring genes encoding ERCC1 and RAD17. Deletion in the last mutant spanned two Chlamydomonas-specific genes with unknown function, demonstrating the utility of this approach for discovering novel factors involved in genome maintenance.     

Polymorphic Sites and the Mechanism of Evolution in Human Mitochondrial DNA

Twelve restriction enzymes were used to screen for the presence or absence of cleavage sites at 441 locations in the mitochondrial DNA of 112 humans from four continents. Cleavage maps were constructed by comparison of DNA fragment sizes with those expected from the published sequence for one human mtDNA. One hundred and sixty-three of the sites were polymorphic, i.e., present in some individuals but absent from others, 278 sites being invariant. These polymorphisms probably result from single base substitutions and occur in all functional regions of the genome.--In 77 cases, it was possible to specify the exact nature and location (within a restriction site) of the mutation responsible for the absence of a restriction site in a known human mtDNA sequence and its presence in another human mtDNA. Fifty-two of these 77 gain mutations occur in genes coding for proteins, 34 being silent and 18 causing amino acid replacements; moreover, nine of the replacements are radical.--Notable also is the anomalous ratio of transitions to transversions required to account for these 77 restriction site differences between the known human mtDNA sequences and other human mtDNAs. This ratio is lower for most groups of restriction sites than has been reported from sequence comparisons of limited parts of the mtDNA genome in closely related mammals, perhaps indicating a special functional role or sensitivity to mutagenesis for palindromic regions containing high levels of guanine and cytosine.--From the genomic distribution of the 163 polymorphic sites, it is inferred that the level of point mutational variability in tRNA and rRNA genes is nearly as high as in protein-coding genes but lower than in noncoding mtDNA. Thus, the functional constraints operating on components of the protein-synthetic apparatus may be lower for mitochondria than for other systems. Furthermore, the mitochondrial genes for tRNAs that recognize four codons are more variable than those recognizing only two codons.--Among the more variable of the human mitochondrial genes coding for proteins is that for subunit 2 of cytochrome oxidase; this polypeptide appears to have been evolving about five times faster in primates than in other mammals. Cytochrome c, a nuclearly encoded protein that interacts directly with the oxidase 2 subunit in electron transport, has also evolved faster in primates than in rodents or ungulates. This example, along with that for the mitochondrial rRNA genes and the nuclear genes coding for mitochondrial ribosomal proteins, provides evidence for coevolution between specific nuclear and mitochondrial genes.     

Hyperthermophiles and the problem of DNA instability

Rates of chemical decomposition of DNA at the optimal growth temperatures of hyperthermophiles seem incongruent with the requirements of accurate genome replication. The peculiar physiology, ecology and phylogeny of hyperthermophiles combine to suggest that these prokaryotes have solved a molecular problem (spontaneous loss of native DNA structure) of a magnitude that well-studied microorganisms do not face. The failure of DNA base composition to correlate with optimal growth temperature among hyperthermophiles provides indirect evidence that other mechanisms maintain their chromosomal DNA in the duplex form. Studies in vitro indicate that DNA primary structure is more difficult to maintain at extremely high temperature than is secondary structure, yet hyperthermophiles exhibit only modest levels of spontaneous mutation. Radiation sensitivity studies also indicate that hyperthermophiles repair their DNA efficiently in vivo , and underlying mechanisms are beginning to be examined. Several enzymes of DNA metabolism from hyperthermophilic archaea exhibit unusual biochemical features that may ultimately prove relevant to DNA repair. However, genomic sequencing results suggest that many DNA repair genes of hyperthermophilic archaea may not be recognized because they are not sufficiently related to those of well-studied organisms.     

Biased distribution of DNA uptake sequences towards genome maintenance genes

Repeated sequence signatures are characteristic features of all genomic DNA. We have made a rigorous search for repeat genomic sequences in the human pathogens Neisseria meningitidis, Neisseria gonorrhoeae and Haemophilus influenzae and found that by far the most frequent 9–10mers residing within coding regions are the DNA uptake sequences (DUS) required for natural genetic transformation. More importantly, we found a significantly higher density of DUS within genes involved in DNA repair, recombination, restriction-modification and replication than in any other annotated gene group in these organisms. Pasteurella multocida also displayed high frequencies of a putative DUS identical to that previously identified in H.influenzae and with a skewed distribution towards genome maintenance genes, indicating that this bacterium might be transformation competent under certain conditions. These results imply that the high frequency of DUS in genome maintenance genes is conserved among phylogenetically divergent species and thus are of significant biological importance. Increased DUS density is expected to enhance DNA uptake and the over-representation of DUS in genome maintenance genes might reflect facilitated recovery of genome preserving functions. For example, transient and beneficial increase in genome instability can be allowed during pathogenesis simply through loss of antimutator genes, since these DUS-containing sequences will be preferentially recovered. Furthermore, uptake of such genes could provide a mechanism for facilitated recovery from DNA damage after genotoxic stress.     

Genomic MRI - a Public Resource for Studying Sequence Patterns within Genomic DNA

Non-coding genomic regions in complex eukaryotes, including intergenic areas, introns, and untranslated segments of exons, are profoundly non-random in their nucleotide composition and consist of a complex mosaic of sequence patterns. These patterns include so-called Mid-Range Inhomogeneity (MRI) regions -- sequences 30-10000 nucleotides in length that are enriched by a particular base or combination of bases (e.g. (G+T)-rich, purine-rich, etc.). MRI regions are associated with unusual (non-B-form) DNA structures that are often involved in regulation of gene expression, recombination, and other genetic processes (Fedorova & Fedorov 2010). The existence of a strong fixation bias within MRI regions against mutations that tend to reduce their sequence inhomogeneity additionally supports the functionality and importance of these genomic sequences (Prakash et al. 2009).Here we demonstrate a freely available Internet resource -- the Genomic MRI program package -- designed for computational analysis of genomic sequences in order to find and characterize various MRI patterns within them (Bechtel et al. 2008). This package also allows generation of randomized sequences with various properties and level of correspondence to the natural input DNA sequences. The main goal of this resource is to facilitate examination of vast regions of non-coding DNA that are still scarcely investigated and await thorough exploration and recognition.     

DNA Repair Mechanisms and the Bypass of DNA Damage in Saccharomyces cerevisiae

DNA repair mechanisms are critical for maintaining the integrity of genomic DNA, and their loss is associated with cancer predisposition syndromes. Studies in Saccharomyces cerevisiae have played a central role in elucidating the highly conserved mechanisms that promote eukaryotic genome stability. This review will focus on repair mechanisms that involve excision of a single strand from duplex DNA with the intact, complementary strand serving as a template to fill the resulting gap. These mechanisms are of two general types: those that remove damage from DNA and those that repair errors made during DNA synthesis. The major DNA-damage repair pathways are base excision repair and nucleotide excision repair, which, in the most simple terms, are distinguished by the extent of single-strand DNA removed together with the lesion. Mistakes made by DNA polymerases are corrected by the mismatch repair pathway, which also corrects mismatches generated when single strands of non-identical duplexes are exchanged during homologous recombination. In addition to the true repair pathways, the postreplication repair pathway allows lesions or structural aberrations that block replicative DNA polymerases to be tolerated. There are two bypass mechanisms: an error-free mechanism that involves a switch to an undamaged template for synthesis past the lesion and an error-prone mechanism that utilizes specialized translesion synthesis DNA polymerases to directly synthesize DNA across the lesion. A high level of functional redundancy exists among the pathways that deal with lesions, which minimizes the detrimental effects of endogenous and exogenous DNA damage.     

Variation in mutation dynamics across the maize genome as a function of regional and flanking base composition

We examine variation in mutation dynamics across a single genome (Zea mays ssp. mays) in relation to regional and flanking base composition using a data set of 10,472 SNPs generated by resequencing 1776 transcribed regions. We report several relationships between flanking base composition and mutation pattern. The A + T content of the two sites immediately flanking the mutation site is correlated with rate, transition bias, and GC --> AT pressure. We also observe a significant CpG effect, or increase in transition rate at CpG sites. At the regional level we find that the strength of the CpG effect is correlated with regional A + T content, ranging from a 1.7-fold increase in transition rate in relatively G + C-rich regions to a 2.6-fold increase in A + T-rich regions. We also observe a relationship between locus A + T content and GC --> AT pressure. This regional effect is in opposition to the influence of the two immediate neighbors in that GC --> AT pressure increases with increasing locus A + T content but decreases with increasing flanking base A + T content and may represent a relationship between genome location and mutation bias. The data indicate multiple context effects on mutations, resulting in significant variation in mutation dynamics across the genome.     

Heterogeneity of nitrogen mustard-induced DNA damage and repair at the level of the gene in Chinese hamster ovary cells

We here present a general method to detect alkylation damage in specific genomic regions. Cells are treated with nitrogen mustard or dimethyl sulfate; the DNA is extracted and restricted, and the parental DNA is separated. Strand breaks are created at sites of N-alkylpurines by neutral depurination followed by alkaline hydrolysis. The DNA is then separated on alkaline agarose gels and transferred, and gene fragments are detected after hybridization with specific probes. Using this approach, we have examined damage formation and repair in the active genes dihydrofolate reductase and adenosine phosphoribosyltransferase, in a fragment containing the inactive c-fos gene and in a nontranscribed region downstream from the dihydrofolate reductase gene in Chinese hamster ovary cells. We find variations in the formation of nitrogen mustard adducts in these different regions. Nitrogen mustard adducts are preferentially repaired from the active genes as compared to the inactive gene and the noncoding region. However, we find no preferential damage or repair in these regions of the N7-methylpurines after dimethyl sulfate damage. Thus, there are significant differences in the repair mechanisms for two alkylating agents; this may implicate that there are important differences in the structural alterations in chromatin invoked by these agents. As a comparison to the studies of adduct levels in specific genomic regions, we have examined the overall genome, average adduct formation, and repair by these agents in the hamster cells. We used alkaline sucrose gradient sedimentation, and also a novel approach: quantitation of the DNA smears stained by ethidium bromide in the alkaline gels (used in the gene-selective repair analysis). Both these techniques gave similar data for adduct formation and repair; there was less initial damage formation and repair in the average genome than in specific genomic regions.     

Glyceraldehyde-3-phosphate dehydrogenase is required for efficient repair of cytotoxic DNA lesions in Escherichia coli

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional protein with diverse biological functions in human cells. In bacteria, moonlighting GAPDH functions have only been described for the secreted protein in pathogens or probiotics. At the intracellular level, we previously reported the interaction of Escherichia coli GAPDH with phosphoglycolate phosphatase, a protein involved in the metabolism of the DNA repair product 2-phosphoglycolate, thus suggesting a putative role of GAPDH in DNA repair processes. Here, we provide evidence that GAPDH is required for the efficient repair of DNA lesions in E. coli. We show that GAPDH-deficient cells are more sensitive to bleomycin or methyl methanesulfonate. In cells challenged with these genotoxic agents, GAPDH deficiency results in reduced cell viability and filamentous growth. In addition, the gapA knockout mutant accumulates a higher number of spontaneous abasic sites and displays higher spontaneous mutation frequencies than the parental strain. Pull-down experiments in different genetic backgrounds show interaction between GAPDH and enzymes of the base excision repair pathway, namely the AP-endonuclease Endo IV and uracil DNA glycosylase. This finding suggests that GAPDH is a component of a protein complex dedicated to the maintenance of genomic DNA integrity. Our results also show interaction of GAPDH with the single-stranded DNA binding protein. This interaction may recruit GAPDH to the repair sites and implicates GAPDH in DNA repair pathways activated by profuse DNA damage, such as homologous recombination or the SOS response.     

Conditional DNA repair mutants enable highly precise genome engineering

Oligonucleotide-mediated multiplex genome engineering is an important tool for bacterial genome editing. The efficient application of this technique requires the inactivation of the endogenous methyl-directed mismatch repair system that in turn leads to a drastically elevated genomic mutation rate and the consequent accumulation of undesired off-target mutations. Here, we present a novel strategy for mismatch repair evasion using temperature-sensitive DNA repair mutants and temporal inactivation of the mismatch repair protein complex in Escherichia coli. Our method relies on the transient suppression of DNA repair during mismatch carrying oligonucleotide integration. Using temperature-sensitive control of methyl-directed mismatch repair protein activity during multiplex genome engineering, we reduced the number of off-target mutations by 85%, concurrently maintaining highly efficient and unbiased allelic replacement.