Review
DNA double-strand break repair from head to tail

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Abstract

DNA double-strand break repair is a complex process that requires multiple enzymatic and structural activities to rejoin or repair the broken DNA ends using one of several repair pathways. These enzymatic and structural activities include end detection, end processing and alignment of DNA ends. Recent structural and functional studies of the DNA double-strand break repair factors Mre11/Rad50, Ku70/80 and Xrcc4 show how these enzymes combine and assemble both enzymatic and structural activities in DNA double-strand break repair.

Introduction

A DNA double-strand break (DSB) is highly cytotoxic DNA damage that disrupts the genomic integrity of a cell. Unrepaired or misrepaired DSBs can kill a cell or lead to chromosome aberrations; thus the prompt and efficient repair of DSBs is fundamental for genomic stability and cancer prevention 1., 2.. DSBs occur as products of ionizing radiation and genotoxic agents, and are often formed during replication of damaged DNA [3]. DNA ends generated by enzymatic activities, however, are also key intermediates in recombination events, such as the generation of antibody and T-cell receptor diversity, meiotic crossing-over and yeast mating type switching 4., 5., 6., and must be processed appropriately.

DSBs are repaired by a variety of different processes, such as homologous recombination (HR), nonhomologous end joining (NHEJ) or single-strand annealing [7]. In all of these processes, DSBs have to be efficiently detected and processed to enable subsequent repair steps. In HR, the end is resected by a nuclease activity to form a 3′ single-stranded (ss) DNA tail. A homology search pairs this tail with the appropriate segment on the sister chromatid to allow strand invasion, homologous recombination and/or DNA repair synthesis, all of which can proceed without loss of a single nucleotide. In NHEJ, the two broken ends must be aligned, processed and religated, and can give rise to chromosomal translocations or other genetic events that change or lose genetic information. To carry out these multiple steps, the DSB repair machineries not only have to possess enzymatic activities, such as DNA end processing activity, but also must exhibit structural activities, for example, to align the DNA ends with the sister chromatids in HR or with other DNA ends in NHEJ. Genetic studies indicate that key players in the cellular response to DSBs are the evolutionarily conserved complex of the Mre11 nuclease and the Rad50 ATPase (denoted the Mre11 complex), and the Ku70/80 heterodimer. In this review, we summarize recent structural and functional progress in our understanding of how the Mre11 complex combines multiple roles to control the initial events of DSB repair and recombination. In addition, we aim to summarize recent results obtained from crystal structures of the Ku70/80 heterodimer and Xrcc4. These structures provide a first view of how DNA ends are recognized and repaired in NHEJ 8••., 9•., 10•..

Section snippets

The Mre11 complex

Homologs of the Mre11 nuclease and the Rad50 ATPase are found in every kingdom of life, suggesting that the Mre11 complex is fundamental for genomic stability [11]. In eukaryotes, the Mre11 complex can contain a third component, which is less conserved and is termed Xrs2 in yeast and Nbs1 in higher eukaryotes 12., 13., 14.. Additional interactions of the Mre11 complex with the breast cancer susceptibility protein BRCA1, a protein of unknown function, RINT-1 and E2F have been reported 15., 16.,

Biochemistry of the Mre11 complex

Biochemical studies showed that Mre11 has ssDNA endonuclease, 3′→5′ double-stranded (ds) DNA exonuclease and hairpin opening activities 23., 26., 45., 46., 47., 48., 49., 50., 51.. The latter two activities are stimulated by or require Rad50 and ATP. All nucleolytic activities of Mre11 require manganese, although DNA binding can also occur in the presence of other divalent metals. Mre11 can bind both ssDNA and dsDNA, and does not need ends for DNA binding [52]. It also has the capability to

Structure of the Mre11 complex

Electron microscopic analysis of proteins from archaebacteria and humans revealed that the Mre11 complex forms an elongated, bipolar structure containing a globular head and a long coiled-coil tail [54••] (Fig. 1). The principal architecture seems to be conserved in bacteria and eukaryotes 55., 56.. The bipolar structure immediately suggests a mechanism for both the enzymatic and structural roles of the complex. The globular head, which represents the complex of Mre11 and the Rad50 ATPase

Mechanism of ATP-dependent DNA processing

The crystal structure of archaeal Mre11 revealed a two-domain protein, consisting of a protein-phosphatase-like domain that contains the nuclease active site and a small capping domain that controls active site access [54••]. The capping domain presumably carries the DNA-binding specificity. The active site of Mre11 is formed by a dimanganese catalytic center. The apparently similar active site geometries of Mre11 and calcineurin-like protein phosphatases suggests that these two enzyme families

DNA end binding in nonhomologous end joining

NHEJ is the predominant repair pathway of DSBs in mammalian cells in G0, G1 and early S phase 61., 62.. In the immune system, DSB intermediates that are created during V(D)J recombination are exclusively repaired by NHEJ [63]. The evolutionarily conserved core NHEJ machinery includes the DNA-end-binding Ku70/80 heterodimer, the accessory factor Xrcc4 and DNA ligaseIV[64].

In the initial steps after a DSB in NHEJ, each DNA end is bound by a Ku heterodimer. Ku recognizes a variety of different DNA

Conclusions and outlook

The recently determined crystal structures of the DNA end detection, alignment and processing factors in HR and NHEJ greatly advanced our understanding of the molecular mechanism governing these steps. However, some important questions remain. Is Mre11 the nuclease responsible for the 5′→3′ DNA end resection before recombination? In both HR and meiosis, the 5′ end has to be degraded to enable subsequent repair steps. Mre11 seems unsuitable to this activity because it degrades DNA ends 3′→5′,

Update

New biochemical and AFM data suggest that the budding yeast Mre11 complex can directly align two broken DNA strands for rejoining [67]. The AFM data show that the globular DNA-binding head of the Mre11 complex can simultaneously bind two ends. These data are consistent with the heterotetrameric model of the Mre11 complex (Fig. 1), which implies the presence of two DNA-binding sites per Mre11 complex. Furthermore, the end alignment action of the Mre11 complex specifically enhances ligation by

References and recommended reading

Papers of particular interest, published within the annual period of review,have been highlighted as:

  • • of special interest

  • •• of outstanding interest

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