Bacterial DNA repair genes and their eukaryotic homologues: 2. Role of bacterial mutator gene homologues in human disease. Overview of nucleotide pool sanitization and mismatch repair systems

Since the discovery of the first E. coli mutator gene, mutT, most of the mutations inducing elevated spontaneous mutation rates could be clearly attributed to defects in DNA repair. MutT turned out to be a pyrophosphohydrolase hydrolyzing 8-oxodGTP, thus preventing its incorporation into DNA and suppresing the occurrence of spontaneous AT-->CG transversions. Most of the bacterial mutator genes appeared to be evolutionarily conserved, and scientists were continuously searching for contribution of DNA repair deficiency in human diseases, especially carcinogenesis. Yet a human MutT homologue--hMTH1 protein--was found to be overexpressed rather than inactivated in many human diseases, including cancer. The interest in DNA repair contribution to human diseases exploded with the observation that germline mutations in mismatch repair (MMR) genes predispose to hereditary non-polyposis colorectal cancer (HNPCC). Despite our continuously growing knowledge about DNA repair we still do not fully understand how the mutator phenotype contributes to specific forms of human diseases.     

Bacterial DNA repair genes and their eukaryotic homologues: 1. Mutations in genes involved in base excision repair (BER) and DNA-end processors and their implication in mutagenesis and human disease

Base excision repair (BER) pathway executed by a complex network of proteins is the major system responsible for the removal of damaged DNA bases and repair of DNA single strand breaks (SSBs) generated by environmental agents, such as certain cancer therapies, or arising spontaneously during cellular metabolism. Both modified DNA bases and SSBs with ends other than 3'-OH and 5'-P are repaired either by replacement of a single or of more nucleotides in the processes called short-patch BER (SP-BER) or long-patch BER (LP-BER), respectively. In contrast to Escherichia coli cells, in human ones, the two BER sub-pathways are operated by different sets of proteins. In this review the selection between SP- and LP-BER and mutations in BER and end-processors genes and their contribution to bacterial mutagenesis and human diseases are considered.     

Bacterial ion channels and their eukaryotic homologues.

Due to the relative ease of obtaining their crystal structures, bacterial ion channels provide a unique opportunity to analyse structure and function of their eukaryotic homologues. This review describes prokaryotic channels whose structures have been determined. These channels are KcsA, a bacterial homologue of eukaryotic potassium channels, MscL, a bacterial mechanosensitive ion channel and ClC0, a prokaryotic homologue of the eukaryotic ClC family of anion-selective channels. General features of their structure and function are described with a special emphasis on the advantages that these channels offer for understanding the properties of their eukaryotic homologues. We present amino-acid sequences of eukaryotic proteins related in their primary sequences to bacterial mechanosensitive channels. The usefulness of bacterial mechanosensitive channels for the studies on general principles of mechanosensation is discussed.     

DNA repeats and homologous recombination: a probable role for DNA methylation in genome stability of eukaryotic cells

Homologous recombination between DNA repeats directly threatens the intact transmission of repeat-rich eukaryotic genomes through mitotic and meiotic cell divisions. Besides several other factors already known, DNA methylation might contribute, in some eukaryotes, to the limitation of crossover events between repeats. A strong inhibitory effect of DNA methylation has now been directly demonstrated, in the filamentous fungus Ascobolus. This therefore reinforces the question of the biological impact of this DNA modification on the recombinational stability of repeat-rich genomes, such as those of mammals.     

The role of DNA helicases and their interaction partners in genome stability and meiotic recombination in plants

DNA helicases are enzymes that are able to unwind DNA by the use of the energy-equivalent ATP. They play essential roles in DNA replication, DNA repair, and DNA recombination in all organisms. As homologous recombination occurs in somatic and meiotic cells, the same proteins may participate in both processes, albeit not necessarily with identical functions. DNA helicases involved in genome stability and meiotic recombination are the focus of this review. The role of these enzymes and their characterized interaction partners in plants will be summarized. Although most factors are conserved in eukaryotes, plant-specific features are becoming apparent. In the RecQ helicase family, Arabidopsis thaliana RECQ4A has been shown before to be the functional homologue of the well-researched baker's yeast Sgs1 and human BLM proteins. It was surprising to find that its interaction partners AtRMI1 and AtTOP3α are absolutely essential for meiotic recombination in plants, where they are central factors of a formerly underappreciated dissolution step of recombination intermediates. In the expanding group of anti-recombinases, future analysis of plant helicases is especially promising. While no FBH1 homologue is present, the Arabidopsis genome contains homologues of both SRS2 and RTEL1. Yeast and mammals, on the other hand. only possess homologues of either one or the other of these helicases. Plants also contain several other classes of helicases that are known from other organisms to be involved in the preservation of genome stability: FANCM is conserved with parts of the human Fanconi anaemia proteins, as are homologues of the Swi2/Snf2 family and of PIF1.     

The mammalian XRCC genes: their roles in DNA repair and genetic stability

Analysis of the XRCC genes has played an important part in understanding mammalian DNA repair processes, especially those involved in double-strand break (DSB) repair. Most of these genes were identified through their ability to correct DNA damage hypersensitivity in rodent cell lines, and they represent components of several different repair pathways including base-excision repair, non-homologous end joining, and homologous recombination. We document the phenotypic effects of mutation of the XRCC genes, and the current state of our knowledge of their functions. In addition to their continuing importance in discovering mechanisms of DNA repair, analysis of the XRCC genes is making a substantial contribution to the understanding of specific human disorders, including cancer.     

DNA repair, genome stability, and aging

Aging can be defined as progressive functional decline and increasing mortality over time. Here, we review evidence linking aging to nuclear DNA lesions: DNA damage accumulates with age, and DNA repair defects can cause phenotypes resembling premature aging. We discuss how cellular DNA damage responses may contribute to manifestations of aging. We review Sir2, a factor linking genomic stability, metabolism, and aging. We conclude with a general discussion of the role of mutant mice in aging research and avenues for future investigation.     

BRCA2 homolog required for proficiency in DNA repair,recombination, and genome stability in Ustilago maydis

In a screen for DNA repair-defective mutants in the fungus Ustilago maydis, a gene encoding a BRCA2 family member, designated here as Brh2, was identified. A brh2 null allele was found to be defective in allelic recombination, meiosis, and repair of gaps and ionizing radiation damage to the same extent as rad51. Frequent marker loss in meiosis and diploid formation suggested that genomic instability was associated with brh2. This notion was confirmed by molecular karyotype analysis, which revealed gross chromosomal alterations associated with brh2. Yeast two-hybrid analysis indicated interaction between Brh2 and Rad51. Recapitulation in U. maydis of defects in DNA repair and genome stability associated with brh2 means that the BRCA2 gene family is more widespread than previously thought.     

Multiple roles of vertebrate REV genes in DNA repair and recombination

In yeast, Rev1, Rev3, and Rev7 are involved in translesion synthesis over various kinds of DNA damage and spontaneous and UV-induced mutagenesis. Here, we disrupted Rev1, Rev3, and Rev7 in the chicken B-lymphocyte line DT40. REV1-/- REV3-/- REV7-/- cells showed spontaneous cell death, chromosomal instability/fragility, and hypersensitivity to various genotoxic treatments as observed in each of the single mutants. Surprisingly, the triple-knockout cells showed a suppressed level of sister chromatid exchanges (SCEs), which may reflect postreplication repair events mediated by homologous recombination, while each single mutant showed an elevated SCE level. Furthermore, REV1-/- cells as well as triple mutants showed a decreased level of immunoglobulin gene conversion, suggesting participation of Rev1 in a recombination-based pathway. The present study gives us a new insight into cooperative function of three Rev molecules and the Polzeta (Rev3-Rev7)-independent role of Rev1 in vertebrate cells.     

Loss of genes for DNA recombination and repair in the reductive genome evolution of thioautotrophic symbionts of Calyptogena clams

Background

Two Calyptogena clam intracellular obligate symbionts, Ca. Vesicomyosocius okutanii (Vok; C. okutanii symbiont) and Ca. Ruthia magnifica (Rma; C. magnifica symbiont), have small genomes (1.02 and 1.16 Mb, respectively) with low G+C contents (31.6% and 34.0%, respectively) and are thought to be in an ongoing stage of reductive genome evolution (RGE). They lack recA and some genes for DNA repair, including mutY. The loss of recA and mutY is thought to contribute to the stabilization of their genome architectures and GC bias, respectively. To understand how these genes were lost from the symbiont genomes, we surveyed these genes in the genomes from 10 other Calyptogena clam symbionts using the polymerase chain reaction (PCR).

Results

Phylogenetic trees reconstructed using concatenated 16S and 23S rRNA gene sequences showed that the symbionts formed two clades, clade I (symbionts of C. kawamurai, C. laubieri, C. kilmeri, C. okutanii and C. soyoae) and clade II (those of C. pacifica, C. fausta, C. nautilei, C. stearnsii, C. magnifica, C. fossajaponica and C. phaseoliformis). recA was detected by PCR with consensus primers for recA in the symbiont of C. phaseoliformis. A detailed homology search revealed a remnant recA in the Rma genome. Using PCR with a newly designed primer set, intact recA or its remnant was detected in clade II symbionts. In clade I symbionts, the recA coding region was found to be mostly deleted. In the Rma genome, a pseudogene of mutY was found. Using PCR with newly designed primer sets, mutY was not found in clade I symbionts but was found in clade II symbionts. The G+C content of 16S and 23S rRNA genes in symbionts lacking mutY was significantly lower than in those with mutY.

Conclusions

The extant Calyptogena clam symbionts in clade II were shown to have recA and mutY or their remnants, while those in clade I did not. The present results indicate that the extant symbionts are losing these genes in RGE, and that the loss of mutY contributed to the GC bias of the genomes during their evolution.     

Base excision repair and its role in maintaining genome stability

For all living organisms, genome stability is important, but is also under constant threat because various environmental and endogenous damaging agents can modify the structural properties of DNA bases. As a defense, organisms have developed different DNA repair pathways. Base excision repair (BER) is the predominant pathway for coping with a broad range of small lesions resulting from oxidation, alkylation, and deamination, which modify individual bases without large effect on the double helix structure. As, in mammalian cells, this damage is estimated to account daily for 10(4) events per cell, the need for BER pathways is unquestionable. The damage-specific removal is carried out by a considerable group of enzymes, designated as DNA glycosylases. Each DNA glycosylase has its unique specificity and many of them are ubiquitous in microorganisms, mammals, and plants. Here, we review the importance of the BER pathway and we focus on the different roles of DNA glycosylases in various organisms.     

The BRCA2-interacting protein DSS1 is vital for DNA repair, recombination, and genome stability in Ustilago maydis

DSS1 encodes a small acidic protein shown in recent structural studies to interact with the DNA binding domain of BRCA2. Here we report that an ortholog of DSS1 is present in Ustilago maydis and associates with Brh2, the BRCA2-related protein, thus recapitulating the protein partnership in this genetically amenable fungus. Mutants of U. maydis deleted of DSS1 are extremely radiation sensitive, deficient in recombination, defective in meiosis, and disturbed in genome stability; these phenotypes mirror previous observations of U. maydis mutants deficient in Brh2 or Rad51. These findings conclusively show that Dss1 constitutes a protein with a significant role in the recombinational repair pathway in U. maydis, and imply that it plays a similar key role in the recombination systems of organisms in which recombinational repair is BRCA2 dependent.     

DNA replication and homologous recombination factors: acting together to maintain genome stability

Genome duplication requires the coordinated action of multiple proteins to ensure a fast replication with high fidelity. These factors form a complex called the Replisome, which is assembled onto the DNA duplex to promote its unwinding and to catalyze the polymerization of two new strands. Key constituents of the Replisome are the Cdc45-Mcm2-7-GINS helicase and the And1-Claspin-Tipin-Tim1 complex, which coordinate DNA unwinding with polymerase alpha-, delta-, and epsilon- dependent DNA polymerization. These factors encounter numerous obstacles, such as endogenous DNA lesions leading to template breakage and complex structures arising from intrinsic features of specific DNA sequences. To overcome these roadblocks, homologous recombination DNA repair factors, such as Rad51 and the Mre11-Rad50-Nbs1 complex, are required to ensure complete and faithful replication. Consistent with this notion, many of the genes involved in this process result in lethal phenotypes when inactivated in organisms with complex and large genomes. Here, we summarize the architectural and functional properties of the Replisome and propose a unified view of DNA replication and repair processes.     

The role of the human SWI5-MEI5 complex in homologous recombination repair

The Swi5-Mei5 complex and its homologues are involved in specialized recombination pathways in budding and fission yeasts. Although the fission yeast homologue Swi5-Sfr1 is critical for homologous recombination repair, the budding yeast counterpart Sae3-Mei5 is meiosis-specific, interacts with Dmc1, and promotes assembly of Dmc1 on meiotic chromosomes. Here, we identify and characterize the human SWI5-MEI5 (C9orf119-C10orf78) complex. We showed that SWI5 and MEI5 form a stable complex in vitro and in vivo. The C-terminal Swi5 domain of SWI5 and the middle coiled-coil region of MEI5 dictate this conserved interaction. In addition, SWI5-MEI5 directly interacts with RAD51 in vitro. Depletion of SWI5 or MEI5 in human cells causes defects in homologous recombination repair. Finally, SWI5- or MEI5-depleted cells display enhanced sensitivity to ionizing radiation, consistent with the role of this complex in HR repair. Our results suggest that human SWI5-MEI5 has an evolutionarily conserved function in homologous recombination repair.     

Coordination of DNA replication and recombination activities in the maintenance of genome stability

Across the evolutionary spectrum, living organisms depend on high-fidelity DNA replication and recombination mechanisms to maintain genome stability and thus to avoid mutation and disease. The repair of severe lesions in the DNA such as double-strand breaks or stalled replication forks requires the coordinated activities of both the homologous recombination (HR) and DNA replication machineries. Growing evidence indicates that so-called "accessory proteins" in both systems are essential for the effective coupling of recombination to replication which is necessary to restore genome integrity following severe DNA damage. In this article we review the major processes of homology-directed DNA repair (HDR), including the double Holliday Junction (dHJ), synthesis-dependent strand annealing (SDSA), break-induced replication (BIR), and error-free lesion bypass pathways. Each of these pathways involves the coupling of a HR event to DNA synthesis. We highlight two major classes of accessory proteins in recombination and replication that facilitate HDR: Recombination mediator proteins exemplified by T4 UvsY, Saccharomyces cerevisiae Rad52, and human BRCA2; and DNA helicases/translocases exemplified by T4 Gp41/Gp59, E. coli DnaB and PriA, and eukaryotic Mcm2-7, Rad54, and Mph1. We illustrate how these factors help to direct the flow of DNA and protein-DNA intermediates on the pathway from a double-strand break or stalled replication fork to a high-fidelity recombination-dependent replication apparatus that can accurately repair the damage.     

Securing genome stability by orchestrating DNA repair: removal of radiation-induced clustered lesions in DNA

In addition to double- and single-strand DNA breaks and isolated base modifications, ionizing radiation induces clustered DNA damage, which contains two or more lesions closely spaced within about two helical turns on opposite DNA strands. Post-irradiation repair of single-base lesions is routinely performed by base excision repair and a DNA strand break is involved as an intermediate. Simultaneous processing of lesions on opposite DNA strands may generate double-strand DNA breaks and enhance nonhomologous end joining, which frequently results in the formation of deletions. Recent studies support the possibility that the mechanism of base excision repair contributes to genome stability by diminishing the formation of double-strand DNA breaks during processing of clustered lesions.