Recruitment of the recombinational repair machinery to a DNA double-strand break in yeast

Repair of DNA double-strand breaks (DSBs) by homologous recombination requires members of the RAD52 epistasis group. Here we use chromatin immunoprecipitation (ChIP) to examine the temporal order of recruitment of Rad51p, Rad52p, Rad54p, Rad55p, and RPA to a single, induced DSB in yeast. Our results suggest a sequential, interdependent assembly of Rad proteins adjacent to the DSB initiated by binding of Rad51p. ChIP time courses from various mutant strains and additional biochemical studies suggest that Rad52p, Rad55p, and Rad54p each help promote the formation and/or stabilization of the Rad51p nucleoprotein filament. We also find that all four Rad proteins associate with homologous donor sequences during strand invasion. These studies provide a near comprehensive view of the molecular events required for the in vivo assembly of a functional Rad51p presynaptic filament.     

Structure and function of the double-strand break repair machinery

The discovery of the recA gene toward the middle of the 20th century sparked work in perhaps one of the most biochemically and biophysically intriguing systems of DNA repair-homologous recombination. The inner workings of this system, in particular those of the more complex eukaryotes, have been and in many ways remain mysterious. Yet at the turn of this century, a wealth of structural and genetic results has unveiled a detailed picture of the roles, relationships, and mechanics of interacting homologous recombination proteins. Here we focus on the predominant questions addressed by these exciting 21st century structural results-from detection of broken DNA ends to coordination of pathway progression. The emerging structural view of double-strand break repair, therefore, reveals the molecular basis both for functions specific to DNA recombination and for general features characterizing DNA repair processes.     

DNA double-strand break repair: from mechanistic understanding to cancer treatment

Accurate repair of DNA double-strand breaks is essential to life. Indeed, defective DNA double-strand break repair can lead to toxicity and large scale sequence rearrangements that cause cancer and promote premature aging. Here, we highlight the two major repair systems for handling DNA double-strand breaks: homologous recombination and non-homologous end joining. To clarify recombination mechanisms, we present animations that illustrate DNA strand movements. In addition to describing how these pathways operate, we also describe why appropriate pathway choice is critical to genomic stability, and we summarize key pathway control features related to cell cycle checkpoint and apoptosis signaling. Importantly, recent progress in delineating the effects of specific defects in repair and checkpoint control has helped to explain several disease phenotypes, including cancer and premature aging. Improved understanding of these pathways has also sparked development of novel chemotherapeutic strategies that kill tumors with increased specificity and efficacy. This review aims to provide a foundational understanding of how the homologous recombination and non-homologous end joining pathways operate, and to demonstrate how a better understanding of these processes has advanced both our understanding of the underlying causes of cancer and our ability to innovate novel cancer treatment strategies.     

Chromatin dynamics in DNA double-strand break repair

DNA double-strand breaks (DSBs) occur in the context of a highly organized chromatin environment and are, thus, a significant threat to the epigenomic integrity of eukaryotic cells. Changes in break-proximal chromatin structure are thought to be a prerequisite for efficient DNA repair and may help protect the structural integrity of the nucleus. Unlike most bona fide DNA repair factors, chromatin influences the repair process at several levels: the existing chromatin context at the site of damage directly affects the access and kinetics of the repair machinery; DSB induced chromatin modifications influence the choice of repair factors, thereby modulating repair outcome; lastly, DNA damage can have a significant impact on chromatin beyond the site of damage. We will discuss recent findings that highlight both the complexity and importance of dynamic and tightly orchestrated chromatin reorganization to ensure efficient DSB repair and nuclear integrity. This article is part of a Special Issue entitled: Chromatin in time and space.     

Molecular mechanisms of DNA double-strand break repair

DNA double-strand breaks (DSBs) are major threats to the genomic integrity of cells. If not taken care of properly, they can cause chromosome fragmentation, loss and translocation, possibly resulting in carcinogenesis. Upon DSB formation, cell-cycle checkpoints are triggered and multiple DSB repair pathways can be activated. Recent research on the Nijmegen breakage syndrome, which predisposes patients to cancer, suggests a direct link between activation of cell-cycle checkpoints and DSB repair. Furthermore, the biochemical activities of proteins involved in the two major DSB repair pathways, homologous recombination and DNA end-joining, are now beginning to emerge. This review discusses these new findings and their implications for the mechanisms of DSB repair.     

Xrcc1-dependent and Ku-dependent DNA double-strand break repair kinetics in Arabidopsis plants

Double-strand breakage (DSB) of DNA involves loss of information on the two strands of the DNA fibre and thus cannot be repaired by simple copying of the complementary strand which is possible with single-strand DNA damage. Homologous recombination (HR) can precisely repair DSB using another copy of the genome as template and non-homologous recombination (NHR) permits repair of DSB with little or no dependence on DNA sequence homology. In addition to the well-characterised Ku-dependent non-homologous end-joining (NHEJ) pathway, much recent attention has been focused on Ku-independent NHR. The complex interrelationships and regulation of NHR pathways remain poorly understood, even more so in the case of plants, and we present here an analysis of Ku-dependent and Ku-independent repair of DSB in Arabidopsis thaliana. We have characterised an Arabidopsis xrcc1 mutant and developed quantitative analysis of the kinetics of appearance and loss of γ-H2AX foci as a tool to measure DSB repair in dividing root tip cells of γ-irradiated plants in vivo. This approach has permitted determination of DSB repair kinetics in planta following a short pulse of γ-irradiation, establishing the existence of a Ku-independent, Xrcc1-dependent DSB repair pathway. Furthermore, our data show a role for Ku80 during the first minutes post-irradiation and that Xrcc1 also plays such a role, but only in the absence of Ku. The importance of Xrcc1 is, however, clearly visible at later times in the presence of Ku, showing that alternative end-joining plays an important role in DSB repair even in the presence of active NHEJ.     

RecG helicase promotes DNA double-strand break repair

Double-strand breaks pose a major threat to the genome and must be repaired accurately if structural and functional integrity are to be preserved. This is usually achieved via homologous recombination, which enables the ends of a broken DNA molecule to engage an intact duplex and prime synthesis of the DNA needed for repair. In Escherichia coli, repair relies on the RecBCD and RecA proteins, the combined ability of which to initiate recombination and form joint-molecule intermediates is well understood. To shed light on subsequent events, we exploited the I-SceI homing endonuclease of yeast to make breaks at I-SceI cleavage sites engineered into the chromosome. We show that survival depends on RecA and RecBCD, and that subsequent events can proceed via either of two pathways, one dependent on the RuvABC Holliday junction resolvase and the other on RecG helicase. Both pathways rely on PriA, presumably to facilitate DNA replication. We discuss the possibility that classical Holliday junctions may not be essential intermediates in repair and consider alternative pathways for RecG-dependent separation of joint molecules formed by RecA.     

DNA double-strand break repair by homologous recombination

The induction of double-strand breaks (DSBs) in DNA by exposure to DNA damaging agents, or as intermediates in normal cellular processes, constitutes a severe threat for the integrity of the genome. If not properly repaired, DSBs may result in chromosomal aberrations, which, in turn, can lead to cell death or to uncontrolled cell growth. To maintain the integrity of the genome, multiple pathways for the repair of DSBs have evolved during evolution: homologous recombination (HR), non-homologous end joining (NHEJ) and single-strand annealing (SSA). HR has the potential to lead to accurate repair of DSBs, whereas NHEJ and SSA are essentially mutagenic. In yeast, DSBs are primarily repaired via high-fidelity repair of DSBs mediated by HR, whereas in higher eukaryotes, both HR and NHEJ are important. In this review, we focus on the functional conservation of HR from fungi to mammals and on the role of the individual proteins in this process.     

DNA double-strand break repair and chromosome translocations

Translocations are genetic aberrations that occur when a broken fragment of a chromosome is erroneously rejoined to another chromosome. The initial event in the creation of a translocation is the formation of a DNA double-strand break (DSB), which can be induced both under physiological situations, such as during the development of the immune system, or by exogenous DNA damaging agents. Two major repair pathways exist in cells that repair DSBs as they arise, namely homologous recombination, and non-homologous end-joining. In some situations these pathways can function inappropriately and rejoin ends incorrectly to produce genomic rearrangements, including translocations. Translocations have been implicated in cancer because of their ability to activate oncogenes. Due to selection at the level of the DNA, the cell, and the tissue certain forms of cancer are associated with specific translocations that can be used as a tool for diagnosis and prognosis of these cancers.     

DNA double-strand break repair within heterochromatic regions

DNA DSBs (double-strand breaks) represent a critical lesion for a cell, with misrepair being potentially as harmful as lack of repair. In mammalian cells, DSBs are predominantly repaired by non-homologous end-joining or homologous recombination. The kinetics of repair of DSBs can differ widely, and recent studies have shown that the higher-order chromatin structure can dramatically affect the pathway utilized, the rate of repair and the genetic factors required for repair. Studies of the repair of DSBs arising within heterochromatic DNA regions have provided insight into the constraints that higher-order chromatin structure poses on repair and the processing that is uniquely required for the repair of such DSBs. In the present paper, we provide an overview of our current understanding of the process of heterochromatic DSB repair in mammalian cells and consider the evolutionary conservation of the processes.     

DNA double-strand break repair by homologous recombination

DNA double-strand breaks (DSB) are presumed to be the most deleterious DNA lesions as they disrupt both DNA strands. Homologous recombination (HR), single-strand annealing, and non-homologous end-joining are considered to be the pathways for repairing DSB. In this review, we focus on DSB repair by HR. The proteins involved in this process as well as the interactions among them are summarized and characterized. The main emphasis is on eukaryotic cells, particularly the budding yeast Saccharomyces cerevisiae and mammals. Only the RAD52 epistasis group proteins are included.     

DNA双链断裂修复的选择性调控机制

在各种DNA损伤中,DNA双链断裂(double-strand break,DSB)是最为严重的一种,快速准确地修复DSB对维持基因组稳定性起着至关重要的作用。真核生物细胞通过一系列复杂的信号转导途径激活对DSB的修复,其中最为重要的是同源重组和非同源末端连接机制。最近的研究表明,这两种方式在DSB修复的早期是相互竞争的关系,其选择在很大程度上受到53BP1及同源蛋白质的调控。将讨论53BP1作为DSB修复途径的核心因子,在染色质水平整合BRCA1、Ct IP等修复因子和多种组蛋白修饰构成的信号途径,介导同源重组和非同源末端连接通路选择的分子机制。     

Species-specific double-strand break repair and genome evolution in plants

Even closely related eukaryotic species may differ drastically in genome size. While insertion of retroelements represents a major source of genome enlargement, the mechanism mediating species- specific deletions is fairly obscure. We analyzed the formation of deletions during double-strand break (DSB) repair in Arabidopsis thaliana and tobacco, two dicotyledonous plant species differing >20-fold in genome size. DSBs were induced by the rare cutting restriction endonuclease I-SCE:I and deletions were identified by loss of function of a negative selectable marker gene containing an I-SCE:I site. Whereas the partial use of micro-homologies in junction formation was similar in both species, in tobacco 40% of the deletions were accompanied by insertions. No insertions could be detected in Arabidopsis , where larger deletions were more frequent, indicating a putative inverse correlation between genome size and the average length of deletions. Such a correlation has been postulated before by a theoretical study on the evolution of related insect genomes and our study now identifies a possible molecular cause for the phenomenon, indicating that species-specific differences in DSB repair might indeed influence genome evolution.     

Mapping the genetic landscape of DNA double-strand break repair

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Regulation of DNA double-strand break repair pathway choice

DNA double-strand breaks （DSBs） are critical lesions that can result in cell death or a wide variety of genetic alterations including largeor small-scale deletions, loss of heterozygosity, translocations, and chromosome loss. DSBs are repaired by non-homologous end-joining （NHEJ） and homologous recombination （HR）, and defects in these pathways cause genome instability and promote tumorigenesis. DSBs arise from endogenous sources including reactive oxygen species generated during cellular metabolism, collapsed replication forks, and nucleases, and from exogenous sources including ionizing radiation and chemicals that directly or indirectly damage DNA and are commonly used in cancer therapy. The DSB repair pathways appear to compete for DSBs, but the balance between them differs widely among species, between different cell types of a single species, and during different cell cycle phases of a single cell type. Here we review the regulatory factors that regulate DSB repair by NHEJ and HR in yeast and higher eukaryotes. These factors include regulated expression and phosphorylation of repair proteins, chromatin modulation of repair factor accessibility, and the availability of homologous repair templates. While most DSB repair proteins appear to function exclusively in NHEJ or HR, a number of proteins influence both pathways, including the MRE11/RAD50/NBS1（XRS2） complex, BRCA1, histone H2AX, PARP-1, RAD18, DNA-dependent protein kinase catalytic subunit （DNA-PKcs）, and ATM. DNA-PKcs plays a role in mammalian NHEJ, but it also influences HR through a complex regulatory network that may involve crosstalk with ATM, and the regulation of at least 12 proteins involved in HR that are phosphorylated by DNA-PKcs and/or ATM.     

Cell cycle-regulated centers of DNA double-strand break repair

In eukaryotes, homologous recombination is an important pathway for the repair of DNA double-strand breaks. We have studied this process in living cells in the yeast Saccharomyces cerevisiae using Rad52 as a cell biological marker. In response to DNA damage, Rad52 redistributes itself and forms foci specifically during S phase. We have shown previously that Rad52 foci are centers of DNA repair where multiple DNA double-strand breaks colocalize. Here we report a correlation between the timing of Rad52 focus formation and modification of the Rad52 protein. In addition, we show that the two ends of a double-strand break are held tightly together in the majority of cells. Interestingly, in a small but significant fraction of the S phase cells, the two ends of a break separate suggesting that mechanisms exist to reassociate and align these ends for proper DNA repair.     

Analysis of DNA double-strand break repair pathways in mice

During the last years significant new insights have been gained into the mechanism and biological relevance of DNA double-strand break (DSB) repair in relation to genome stability. DSBs are a highly toxic DNA lesion, because they can lead to chromosome fragmentation, loss and translocations, eventually resulting in cancer. DSBs can be induced by cellular processes such as V(D)J recombination or DNA replication. They can also be introduced by exogenous agents DNA damaging agents such as ionizing radiation or mitomycin C. During evolution several pathways have evolved for the repair of these DSBs. The most important DSB repair mechanisms in mammalian cells are nonhomologous end-joining and homologous recombination. By using an undamaged repair template, homologous recombination ensures accurate DSB repair, whereas the untemplated nonhomologous end-joining pathway does not. Although both pathways are active in mammals, the relative contribution of the two repair pathways to genome stability differs in the different cell types. Given the potential differences in repair fidelity, it is of interest to determine the relative contribution of homologous recombination and nonhomologous end-joining to DSB repair. In this review, we focus on the biological relevance of DSB repair in mammalian cells and the potential overlap between nonhomologous end-joining and homologous recombination in different tissues.