Surveillance mechanisms monitoring chromosome breaks during mitosis and meiosis

DNA double-strand breaks (DSBs) are highly hazardous for genome integrity, because failure to repair them can lead to genome rearrangements or chromosome loss. They can arise at unpredictable locations as a consequence of DNA damage during both the mitotic and the meiotic cell cycle or in a programmed manner during meiosis. Cellular response to accidental or programmed DSBs involves highly conserved surveillance mechanisms, called DNA damage checkpoint and recombination checkpoint, which coordinate DSB repair with mitotic or meiotic cell cycle progression, respectively. Although these protective signal-transduction pathways share several upstream components, activation of the recombination checkpoint requires meiosis-specific proteins. These proteins are structural components of the meiotic chromosomes, indicating that the system monitoring programmed meiotic DSBs is an integral part of the chromosome structure formed during meiosis.     

DNA double-strand breaks in meiosis: Checking their formation, processing and repair

DNA double-strand breaks (DSBs) are highly hazardous for genome integrity, but meiotic cells deliberately introduce them into their genome in order to initiate homologous recombination, which ensures proper homologous chromosome segregation. To minimize the risk of deleterious effects, meiotic DSB formation, processing and repair are tightly regulated in order to occur only at the right time and place. Furthermore, a highly conserved signal-transduction pathway, called meiotic recombination checkpoint, coordinates DSB repair with meiotic progression and promotes meiotic recombination.     

Double-stranded DNA breaks and gene functions in recombination and meiosis

Meiotic prophase I is a long and complex phase. Homologous recombination is an important process that occurs between homologous chromosomes during meiotic prophase I. Formation of chiasmata, which hold homologous chromosomes together until the metaphase I to anaphase I transition, is critical for proper chromosome segregation. Recent studies have suggested that the SPO 11 proteins have conserved functions in a number of organisms in generating sites of double-stranded DNA breaks （DSBs） that are thought to be the starting points of homologous recombination. Processing of these sites of DSBs requires the function of RecA homologs, such as RAD5 1, DMC 1, and others, as suggested by mutant studies; thus the failure to repair these meiotic DSBs results in abnormal chromosomal alternations, leading to disrupted meiosis. Recent discoveries on the functions of these RecA homologs have improved the understanding of the mechanisms underlying meiotic homologous recombination.     

Novel and diverse functions of the DNA mismatch repair family in mammalian meiosis and recombination

The mismatch repair (MMR) family is a highly conserved group of proteins that function in genome stabilization and mutation avoidance. Their role has been particularly well studied in the context of DNA repair following replication errors, and disruption of these processes results in characteristic microsatellite instability, repair defects and, in mammals, susceptibility to cancer. An additional role in meiotic recombination has been described for several family members, as revealed by extensive studies in yeast. More recently, the role of the mammalian MMR family in meiotic progression has been elucidated by the phenotypic analysis of mice harboring targeted mutations in the genes encoding several MMR family members. This review will discuss the phenotypes of the various mutant mouse lines and, drawing from our knowledge of MMR function in yeast meiosis and in somatic cell repair, will attempt to elucidate the significance of MMR activity in mouse germ cells. These studies highlight the importance of comparative analysis of MMR orthologs across species, and also underscore distinct sexually dimorphic characteristics of mammalian recombination and meiosis.     

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.     

RecQ helicases in DNA double strand break repair and telomere maintenance

Organisms are constantly exposed to various environmental insults which could adversely affect the stability of their genome. To protect their genomes against the harmful effect of these environmental insults, organisms have evolved highly diverse and efficient repair mechanisms. Defective DNA repair processes can lead to various kinds of chromosomal and developmental abnormalities. RecQ helicases are a family of evolutionarily conserved, DNA unwinding proteins which are actively engaged in various DNA metabolic processes, telomere maintenance and genome stability. Bacteria and lower eukaryotes, like yeast, have only one RecQ homolog, whereas higher eukaryotes including humans possess multiple RecQ helicases. These multiple RecQ helicases have redundant and/or non-redundant functions depending on the types of DNA damage and DNA repair pathways. Humans have five different RecQ helicases and defects in three of them cause autosomal recessive diseases leading to various kinds of cancer predisposition and/or aging phenotypes. Emerging evidence also suggests that the RecQ helicases have important roles in telomere maintenance. This review mainly focuses on recent knowledge about the roles of RecQ helicases in DNA double strand break repair and telomere maintenance which are important in preserving genome integrity.     

Mouse TEX15 is essential for DNA double-strand break repair and chromosomal synapsis during male meiosis

During meiosis, homologous chromosomes undergo synapsis and recombination. We identify TEX15 as a novel protein that is required for chromosomal synapsis and meiotic recombination. Loss of TEX15 function in mice causes early meiotic arrest in males but not in females. Specifically, TEX15-deficient spermatocytes exhibit a failure in chromosomal synapsis. In mutant spermatocytes, DNA double-strand breaks (DSBs) are formed, but localization of the recombination proteins RAD51 and DMC1 to meiotic chromosomes is severely impaired. Based on these data, we propose that TEX15 regulates the loading of DNA repair proteins onto sites of DSBs and, thus, its absence causes a failure in meiotic recombination.     

Functional Validation of Rare Human Genetic Variants Involved in Homologous Recombination Using Saccharomyces cerevisiae

Systems for the repair of DNA double-strand breaks (DSBs) are necessary to maintain genome integrity and normal functionality of cells in all organisms. Homologous recombination (HR) plays an important role in repairing accidental and programmed DSBs in mitotic and meiotic cells, respectively. Failure to repair these DSBs causes genome instability and can induce tumorigenesis. Rad51 and Rad52 are two key proteins in homologous pairing and strand exchange during DSB-induced HR; both are highly conserved in eukaryotes. In this study, we analyzed pathogenic single nucleotide polymorphisms (SNPs) in human RAD51 and RAD52 using the Polymorphism Phenotyping (PolyPhen) and Sorting Intolerant from Tolerant (SIFT) algorithms and observed the effect of mutations in highly conserved domains of RAD51 and RAD52 on DNA damage repair in a Saccharomyces cerevisiae-based system. We identified a number of rad51 and rad52 alleles that exhibited severe DNA repair defects. The functionally inactive SNPs were located near ATPase active site of Rad51 and the DNA binding domain of Rad52. The rad51-F317I, rad52-R52W, and rad52-G107C mutations conferred hypersensitivity to methyl methane sulfonate (MMS)-induced DNA damage and were defective in HR-mediated DSB repair. Our study provides a new approach for detecting functional and loss-of-function genetic polymorphisms and for identifying causal variants in human DNA repair genes that contribute to the initiation or progression of cancer.     

Variation and evolution of meiosis

Meiosis arose in the evolution of primitive unicellular organisms as a part of sexual process. One type of meiosis, the so-called classical type, predominates in all kingdoms of eukaryotes. Meiosis is controlled by hundreds of genes, both shared with mitosis and specifically meiotic ones. In a wide range of taxa, which in some cases include kingdoms, meiotic genes and features obey Vavilov's law of homologous variation series. Synaptonemal complexes (SCs) temporarily binding homologous chromosomes at prophase I, ensure precise and equal crossing over and interference. SC proteins have 60-80% homology within the class of mammals but differ from the corresponding proteins in fungi and plants. Thus, nonhomologous SC proteins perform similar functions in different taxa. Some recombination enzymes in fungi and insects have common epitopes. The molecular mechanism of recombination is inherited by eukaryotes from prokaryotes and operates in special compartments: SC recombination nodules. Chiasmata, i.e., physical crossovers of nonsister chromatids, are preserved in bivalents until metaphase I due to local cohesion of sister chromatids in the remaining SC fragments. Owing to chiasmata, homologous chromosomes participate in meiosis I in pairs rather than individually, which, along with unipolarity of kinetochores (only in meiosis 1), ensures segregation of homologous chromosomes. The appearance of SC and chiasmata played a key role in the evolution of unicellular organisms since it promoted the development of a progressive type of meiosis. Some lower eukaryotes retain primitive meiosis types. These primitive modes of meiosis also occur in the sex of some insects that is heterozygous for sex chromosomes. I suggest an explanation for these cases. Mutations at meiotic genes impair meiosis; however, due to the preservation of archaic meiotic genes in the genotype, bypass metabolic pathways arise, which provide partial rescue of the traits damaged by mutations. Individual blocks of genetic program of meiotic regulation have probably evolved independently.     

Meiosis,recombination and chromosomes: a review of gene isolation and fluorescent in situ hybridization data in plants

Evidence is now increasing that many functions and processes of meiotic genes are similar in yeast and higher eukaryotes. However, there are significant differences and, most notably, yeast has considerably higher recombination frequencies than higher eukaryotes, different cross-over interference and possibly more than one pathway for recombination, one late and one early. Other significant events are the timing of double-strand breaks (induced by Spo11) that could be either cause or consequence of homologous chromosome synapsis and SC formation depending on the organisms, yeast plants and mammals versus Drosophila melanogaster and Caenorhabditis elegans. Many plant homologues and heterologues to meiotic genes of yeast and other organisms have now been isolated, in particular in Arabidopsis thaliana, showing that overall recombination genes are very conserved while synaptonemal complex and cohesion proteins are not. In addition to the importance of unravelling the meiotic processes by gene discovery, this review discusses the significance of chromatin packaging, genome organization, and distribution of specific repeated DNA sequences for homologous chromosome cognition and pairing, and the distribution of recombination events along the chromosomes.     

The evolution of meiosis: recruitment and modification of somatic DNA-repair proteins

Several DNA-damage detection and repair mechanisms have evolved to repair double-strand breaks induced by mutagens. Later in evolutionary history, DNA single- and double-strand cuts made possible immune diversity by V(D)J recombination and recombination at meiosis. Such cuts are induced endogenously and are highly regulated and controlled. In meiosis, DNA cuts are essential for the initiation of homologous recombination, and for the formation of joint molecule and crossovers. Many proteins that function during somatic DNA-damage detection and repair are also active during homologous recombination. However, their meiotic functions may be altered from their somatic roles through localization, posttranslational modifications and/or interactions with meiosis-specific proteins. Presumably, somatic repair functions and meiotic recombination diverged during evolution, resulting in adaptations specific to sexual reproduction. (c) 2005 Wiley Periodicals, Inc.     

The RecQ gene family in plants

RecQ helicases are conserved throughout all kingdoms of life regarding their overall structure and function. They are 3'-5' DNA helicases resolving different recombinogenic DNA structures. The RecQ helicases are key factors in a number of DNA repair and recombination pathways involved in the maintenance of genome integrity. In eukaryotes the number of RecQ genes and the structure of RecQ proteins vary strongly between organisms. Therefore, they have been named RecQ-like genes. Knockouts of several RecQ-like genes cause severe diseases in animals or harmful cellular phenotypes in yeast. Until now the largest number of RecQ-like genes per organism has been found in plants. Arabidopsis and rice possess seven different RecQ-like genes each. In the almost completely sequenced genome of the moss Physcomitrella patens at least five RecQ-like genes are present. One of the major present and future research aims is to define putative plant-specific functions and to assign their roles in DNA repair and recombination pathways in relation to RecQ genes from other eukaryotes. Regarding their intron positions, the structures of six RecQ-like genes of dicots and monocots are virtually identical indicating a conservation over a time scale of 150 million years. In contrast to other eukaryotes one gene (RecQsim) exists exclusively in plants. It possesses an interrupted helicase domain but nevertheless seems to have maintained the RecQ function. Owing to a recent gene duplication besides the AtRecQl4A gene an additional RecQ-like gene (AtRecQl4B) exists in the Brassicaceae only. Genetic studies indicate that a AtRecQl4A knockout results in sensitivity to mutagens as well as an hyper-recombination phenotype. Since AtRecQl4B was still present, both genes must have non-redundant roles. Analysis of plant RecQ-like genes will not only increase the knowledge on DNA repair and recombination, but also on the evolution and radiation of protein families.     

Differential effect of the overexpression of Rad2/XPG family endonucleases on genome integrity in yeast and human cells

Eukaryotic cells possess several DNA endonucleases that are necessary to complete different steps in DNA metabolism. Rad2/XPG and Rad27/FEN1 belong to a group of evolutionary conserved proteins that constitute the Rad2 family. Given the important roles carried out by these nucleases in DNA repair and their capacity to create DNA breaks, we have investigated the effect that in vivo imbalance of these nucleases and others of the family have on genome integrity and cell proliferation. We show that overexpression of these nucleases causes genetic instability in both yeast and human cells. Interestingly, the type of recombination event and DNA damage induced suggest specific modes and timing of action of each nuclease that are beyond their known DNA repair function and are critical to preserve genome integrity. In addition to identifying new sources of genome instability, a hallmark of cancer cells, this study provides new genetic tools for studies of genome dynamics.     

Homologs of MutS and MutL during mammalian meiosis

In eukaryotes, homologs of the Escherichia coli MutS and MutL proteins are crucial for both meiotic recombination and post-replicative DNA mismatch repair. Both pathways require the formation of a MutS homolog complex which interacts with a second heterodimer, composed of two MutL homologs. During mammalian meiosis, it is likely that chromosome synapsis requires the presence of a MSH4-MSH5 heterodimer. PMS2, a MutL homolog, seems to play an important role in this process. A MSH4-MSH5 heterodimer is also likely present later with other MutL homologs (MLH1 and MLH3) and is involved in the crossing-over process. The phenotype of msh4-/- mutant mice and MSH4 immunolocalization on meiotic chromosomes suggest that MSH4 has an early function in mammalian meiotic recombination. Both MSH4 and PMS2 directly interact with the RAD51 DNA strand exchange protein. In addition, MSH4 and RAD51 proteins co-localize on mouse meiotic chromosome cores. These results suggest that MSH4 and its partners could act, just after strand exchange promoted by RAD51, to check the homology of DNA heteroduplexes.     

Unique invasions and resolutions: DNA repair proteins in meiotic recombination in Drosophila melanogaster

To ensure the accurate disjunction of homologous chromosomes during meiosis, most eukaryotes rely on physical connections called chiasmata, which form at sites of crossing over. In the absence of crossing over, homologs may segregate randomly, resulting in high frequencies of aneuploid gametes. The process of meiotic recombination poses unique problems for the cell that must be overcome to ensure normal disjunction of homologous chromosomes. How is it ensured that crossovers occur between homologous chromosomes, rather than between sister chromatids? What determines the number and location of crossovers? The functions of DNA repair proteins hold some of the answers to these questions. In this review, we discuss DNA repair proteins that function in meiotic recombination in Drosophila melanogaster. We emphasize the processes of strand invasion and Holliday junction resolution in order to shed light on the questions raised above. Also, we compare the variety of ways several eukaryotes perform these processes and the different proteins they require.     

Role of RAD52 Epistasis Group Genes in Homologous Recombination and Double-Strand Break Repair

The process of homologous recombination is a major DNA repair pathway that operates on DNA double-strand breaks, and possibly other kinds of DNA lesions, to promote error-free repair. Central to the process of homologous recombination are the RAD52 group genes (RAD50, RAD51, RAD52, RAD54, RDH54/TID1, RAD55, RAD57, RAD59, MRE11, and XRS2), most of which were identified by their requirement for the repair of ionizing-radiation-induced DNA damage in Saccharomyces cerevisiae. The Rad52 group proteins are highly conserved among eukaryotes, and Rad51, Mre11, and Rad50 are also conserved in prokaryotes and archaea. Recent studies showing defects in homologous recombination and double-strand break repair in several human cancer-prone syndromes have emphasized the importance of this repair pathway in maintaining genome integrity. Although sensitivity to ionizing radiation is a universal feature of rad52 group mutants, the mutants show considerable heterogeneity in different assays for recombinational repair of double-strand breaks and spontaneous mitotic recombination. Herein, I provide an overview of recent biochemical and structural analyses of the Rad52 group proteins and discuss how this information can be incorporated into genetic studies of recombination.     

Role of RAD52 epistasis group genes in homologous recombination and double-strand break repair.

The process of homologous recombination is a major DNA repair pathway that operates on DNA double-strand breaks, and possibly other kinds of DNA lesions, to promote error-free repair. Central to the process of homologous recombination are the RAD52 group genes (RAD50, RAD51, RAD52, RAD54, RDH54/TID1, RAD55, RAD57, RAD59, MRE11, and XRS2), most of which were identified by their requirement for the repair of ionizing-radiation-induced DNA damage in Saccharomyces cerevisiae. The Rad52 group proteins are highly conserved among eukaryotes, and Rad51, Mre11, and Rad50 are also conserved in prokaryotes and archaea. Recent studies showing defects in homologous recombination and double-strand break repair in several human cancer-prone syndromes have emphasized the importance of this repair pathway in maintaining genome integrity. Although sensitivity to ionizing radiation is a universal feature of rad52 group mutants, the mutants show considerable heterogeneity in different assays for recombinational repair of double-strand breaks and spontaneous mitotic recombination. Herein, I provide an overview of recent biochemical and structural analyses of the Rad52 group proteins and discuss how this information can be incorporated into genetic studies of recombination.     

UvrD helicase suppresses recombination and DNA damage-induced deletions

UvrD, a highly conserved helicase involved in mismatch repair, nucleotide excision repair (NER), and recombinational repair, plays a critical role in maintaining genomic stability and facilitating DNA lesion repair in many prokaryotic species. In this report, we focus on the UvrD homolog in Helicobacter pylori, a genetically diverse organism that lacks many known DNA repair proteins, including those involved in mismatch repair and recombinational repair, and that is noted for high levels of inter- and intragenomic recombination and mutation. H. pylori contains numerous DNA repeats in its compact genome and inhabits an environment rich in DNA-damaging agents that can lead to increased rearrangements between such repeats. We find that H. pylori UvrD functions to repair DNA damage and limit homologous recombination and DNA damage-induced genomic rearrangements between DNA repeats. Our results suggest that UvrD and other NER pathway proteins play a prominent role in maintaining genome integrity, especially after DNA damage; thus, NER may be especially critical in organisms such as H. pylori that face high-level genotoxic stress in vivo.     

Structural maintenance of chromosomes (SMC) proteins, a family of conserved ATPases

The structural maintenance of chromosomes (SMC) proteins are essential for successful chromosome transmission during replication and segregation of the genome in all organisms. SMCs are generally present as single proteins in bacteria, and as at least six distinct proteins in eukaryotes. The proteins range in size from approximately 110 to 170 kDa, and each has five distinct domains: amino- and carboxy-terminal globular domains, which contain sequences characteristic of ATPases, two coiled-coil regions separating the terminal domains and a central flexible hinge. SMC proteins function together with other proteins in a range of chromosomal transactions, including chromosome condensation, sister-chromatid cohesion, recombination, DNA repair and epigenetic silencing of gene expression. Recent studies are beginning to decipher molecular details of how these processes are carried out.     

Caenorhabditis elegans msh-5 is required for both normal and radiation-induced meiotic crossing over but not for completion of meiosis

Crossing over and chiasma formation during Caenorhabditis elegans meiosis require msh-5, which encodes a conserved germline-specific MutS family member. msh-5 mutant oocytes lack chiasmata between homologous chromosomes, and crossover frequencies are severely reduced in both oocyte and spermatocyte meiosis. Artificially induced DNA breaks do not bypass the requirement for msh-5, suggesting that msh-5 functions after the initiation step of meiotic recombination. msh-5 mutants are apparently competent to repair breaks induced during meiosis, but accomplish repair in a way that does not lead to crossovers between homologs. These results combine with data from budding yeast to establish a conserved role for Msh5 proteins in promoting the crossover outcome of meiotic recombination events. Apart from the crossover deficit, progression through meiotic prophase is largely unperturbed in msh-5 mutants. Homologous chromosomes are fully aligned at the pachytene stage, and germ cells survive to complete meiosis and gametogenesis with high efficiency. Our demonstration that artificially induced breaks generate crossovers and chiasmata using the normal meiotic recombination machinery suggests (1) that association of breaks with a preinitiation complex is not a prerequisite for entering the meiotic recombination pathway and (2) that the decision for a subset of recombination events to become crossovers is made after the initiation step.