Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
Analysis of DNA double-strand break repair pathways in mice
Introduction
The integrity of genetic information encoded in DNA is essential for cell survival. Endogenous and exogenous DNA damaging agents are constantly challenging the stability of DNA inside cells. Because a large variety of lesions occur in DNA, it is not surprising that multiple pathways have developed that each repair a subset of these lesions [1]. Among these lesions, DNA double-strand breaks (DSBs) represent a very toxic lesion. DSBs can be produced by several external factors, for example ionizing radiation and radiomimetic drugs. In addition, DSBs arise naturally for various reasons. Meiosis and rearrangement of gene segments (V(D)J recombination) during immune cell development are important physiological processes involving DSB intermediates. Non-programmed DSBs occur during a normal cell cycle in S phase when DNA replication forks stall, for example when the DNA template contains damage. Restarting replication under these conditions can involve generation of a DSB intermediate [2].
The repair of DSBs is critical for maintaining genome stability. If unrepaired, DSBs can lead to cell death. If misrepaired, these DSBs can result in chromosomal rearrangements that can underlie carcinogenesis. Accurate genome duplication is controlled by several checkpoints to prevent cells from initiating DNA replication (G1/S checkpoint), from progressing with replication (intra-S checkpoint) or from going into mitosis (G2/M checkpoint) [3], [4]. The p53 protein has been recognized as an important checkpoint protein, functioning mainly through transcriptional control of target genes that influence multiple response pathways. Cells or mice that are deficient in p53 may fail to undergo cell-cycle arrest or apoptosis in response to conditions that either lead to DNA damage [5], [6] or perturb the cell cycle [7]. Therefore, it is noteworthy that several embryonic lethal phenotypes due to defective DSB repair can be rescued in the absence of p53 [8], [9], [10], [11], [12].
Molecular analyses have revealed that DSB healing can occur through several distinct mechanisms, homologous recombination, nonhomologous end-joining, single-strand annealing, telomere maintenance or cDNA capture [1], [13]. In this review we will focus on the two major DSB repair pathways in mammalian cells, homologous recombination and nonhomologous end-joining. Homologous recombination requires extensive regions of DNA sequence homology and repairs DSBs accurately using information on the undamaged sister chromatid or homologous chromosome. Nonhomologous end-joining uses no or little sequence homology to rejoin broken ends in a manner that need not to be error-free. Recent experiments, using mice with defined mutations in genes involved in homologous recombination and nonhomologous end-joining have revealed that both pathways are active in mammals and essential for DSB repair. Our main focus will be on the relative importance of the DSB repair pathways and in particular on cell type-specific differences in use of these different pathways.
Section snippets
Mechanism of homologous recombination
Homologous recombination can be initiated by processing of DNA ends to produce molecules with 3′ single-stranded tails. Following end resection, single-stranded DNA tails are coated with the single-stranded DNA binding protein replication protein A (RPA). Homologous recombination mediators, such as Rad52 and Brca2, help to load Rad51 on this single-stranded DNA tail replacing RPA [14]. The Rad51 coated single-stranded DNA tail, referred to as nucleoprotein filament, can undergo homology-driven
Mechanism of nonhomologous end-joining
In contrast to homologous recombination, nonhomologous end-joining uses little or no homology at all to repair the break. This pathway is not only used to repair DSBs generated by exogenous DNA damaging agents, such as ionizing radiation, but is also required to process the DSB intermediates that are generated during V(D)J recombination [17]. V(D)J recombination creates specialized DSBs, whose rejoining generates the exon that encodes the variable domain for immunoglobulins and T-cell
Mouse mutants defective in homologous recombination
The most important proteins involved in this pathway are the core homologous recombination proteins, Rad51, Rad52, Rad54, Rad54B, the Rad51 paralogs XRCC2, XRCC3, Rad51B, Rad51C, Rad51D, the Brca proteins and the MRN complex consisting of Mre11, Rad50 and Nbs1. Attempts to assess the biological consequences of defects in homologous recombination proteins have been prevented by the embryonic lethality of many mouse knockouts targeting genes involved in homologous recombination (Table 1). Below
Ku70, Ku80, DNA-PKcs
DNA-PK is a holoenzyme composed of the Ku70/80 heterodimer and a catalytic subunit; DNA-PKcs. DNA-PKcs shows serine/threonine protein kinase activity that is enhanced by a simultaneous association with both Ku70/80 and DNA. Ku was first identified as an autoantigen in the sera of patients with autoimmune disease [105]. The Ku heterodimer is the DNA end-binding component of the DNA-PK holoenzyme. Biochemical studies indicate that Ku and DNA-PKcs do not associate in the absence of DNA termini
Overlapping and specialized roles of homologous recombination and nonhomologous end-joining
Rad54−/− mice provided an excellent opportunity to study the biological significance of the mammalian homologous recombination pathway. In contrast to homozygous disruption of other genes implicated in DSB repair; such as Rad51, Mre11, Brca1 and Brca2, which result in an embryonic lethal phenotype, Rad54−/− mice are viable. Rad54−/− mice display no gross abnormalities and have a normal life expectancy. Therefore, Rad54−/− mice provide the opportunity to study the biological relevance of
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2019, Seminars in Arthritis and RheumatismCitation Excerpt :First, homologous recombination (HR, also termed “homology-directed repair [HDR]) repairs DSBs by transfer of DNA template into its homologous target location in the genome. Despite prior extensive use, targeted modification of genomic DNA by HDR has been limited by relative inefficiency, high rates of random template DNA insertion, and off-target insertions [2,3]. The second and most common form of repair is known as non-homologous end joining (NHEJ).
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2011, Mutation Research - Fundamental and Molecular Mechanisms of MutagenesisCitation Excerpt :In many ways these complex responses are required to generate means of dealing, both with the difficulties associated with DSB repair, as well as with the destabilization DSBs cause to chromatin. The set of complex reactions associated with cellular responses to DSBs is termed the DNA damage response (DDR) and it has been shown to be a key component of genomic stability and cell survival and an essential barrier to the development of cancer [2,8,9]. The DDR specifically responds to DSBs and long stretches of single stranded DNA regions.