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In general the MRE11 and RAD50 proteins are highly conserved between yeast and humans. Both proteins are comparable sizes in the two organisms -150 kDa for RAD50 and -8 0 kDa for MRE11 (Petrini et al., 1995; Dolganov of a!.,

1996). Furthermore, the amino acid sequences of hMRE11 and ScMRE11 share 50% ide n tity in the N -term inal region, w hich encom passes the phosphodiesterase domains, although the C-termini are more divergent (Petrini

et a!., 1995). In contrast, the hRAD50 and ScRAD50 proteins share the highest conservation (again >50% identity) at both the N and 0 termini, with an intermediate portion exhibiting much lower sequence homology (Dolganov et a!.,

1996). This is not surprising considering that the Walker A nucleotide binding motif is located within the N-terminal region of RAD50, the Walker B motif within the C-terminus, and the catalytic domain is formed by interaction of the two terminal regions (Dolganov eta!., 1996; Hopfner et a!., 2000). Whilst the central regions of RAD50 may not exhibit high primary sequence conservation, these domains are predicted to adopt extremely similar coiled-coil structures within both the yeast and human proteins (Dolganov et a!., 1996). Moreover, RAD50 and MRE11 from yeast and humans exhibit very similar biochemical properties including the formation of a highly stable RAD50/MRE11 complex (Johzuka and Ogawa, 1995; Dolganov eta!., 1996).

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Comparatively, the yeast Xrs2 and human NBS1 proteins share very little sequence homology; only 28% identity in the N-terminal 115 amino acids (Carney et al., 1998). As such the two proteins are not true homologues, however the two proteins are the same size (95 kDa) and both are components of the MRE11/RAD50 complex (Carney at a!., 1998; Usui at a!., 1998). Xrs2 and NBS1 are therefore considered functional analogues. NBS1 contains two identifiable motifs at the N-terminus, a fork-head-associated (FHA) domain and a BRCT domain (Varon at a!., 1998). Both motifs are found within other DNA- damage-responsive cell cycle checkpoint proteins, and have been suggested to mediate protein-protein interactions. However, it has been shown that the MRE11-binding domain of NBS1 lies at aa 665 - 693, and the FHA and BRCT domains can be deleted without affecting association with MRE11 (Tauchi at a!.,

2001). In addition, the radiosensitivity of MBS cells was rescued by expression of

NBS1 constructs lacking either the FHA or BRCT domains. This indicates that these domains are not essential for DSB repair.

The biochemical activities of the MRE11/RAD50/NBS1 complex (referred to in future as the M/R/N complex) have been investigated in vitro. Due to the high degree of conservation between the yeast and human MRE11 proteins, it was no surprise when purified MRE11 was found to have 3' - 5' dsDNA exonuclease activity (Pauli and Gellert, 1998). The exonuclease activity of MRE11 was stimulated 3- to 4-fold by association with RAD50. However, both the MRE11/RAD50 complex and MRE11 alone showed similar activities in the presence or absence of ATP. Furthermore, the nuclease activity of MRE11 was

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shown to permit the joining, by DNA ligase I or the XRCC4/DNA ligase IV complex, of DNA fragments carrying non-complementary termini (Pauli and Gellert, 1998; Pauli and Gellert, 2000). The mismatched ends were found to undergo deletions of between 13 and 79 nucleotides and junctions were formed when short sequences of microhomology were revealed. In contrast, both wild type and nuclease-defective MRE11 protein stimulated XRCC4/DNA ligase IV- catalysed joining of complementary termini. This suggests that apart from its nuclease activity, MRE11 might also act in end-joining as a DNA end-bridging factor. In support of this theory, MRE11 was also shown to anneal complementary ssDNA molecules (de Jager etal., 2001a).

In addition to the dsDNA exonuclease and annealing activities, MRE11 and the M/R/X complex also exhibited endonuclease activity on ssDNA and hairpin structures (Pauli and Gellert, 1998; Trujillo etal., 1998). Briefly, MRE11 alone was able to cleave unpaired hairpin loops, with the majority of cuts made one or two nucleotides 3' of the centre of the hairpin (Pauli and Gellert, 1998). In the absence of RAD50 or NBS1, though, MRE11 had little activity on hairpins in which all the bases were paired. Association with NBS1 allowed MRE11 to cleave fully paired hairpins (Pauli and Gellert, 1999). Furthermore, in the presence of ATP, the M/R/N complex exhibited limited unwinding of the DNA duplex allowing cleavage of hairpins at a greater variety of sites. One mechanism in which this hairpin cleavage activity of the M/R/N complex could conceivably be required is in the joining of coding sequences during V(D)J recombination.

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In support of a role for MRE11, RAD50 and NBS1 in DSB repair in mammalian cells, immunofluorescence studies have found that these three proteins colocalise in distinct nuclear foci following ionising radiation (Maser et al., 1997; Carney at a!., 1998). Furthermore, in an innovative piece of work human fibroblasts were exposed to ultrasoft X-rays passed through an irradiation mask consisting of small, evenly spaced strips of gold (Nelms eta l., 1998). Consequently the cell nuclei were irradiated in distinctive stripes, and the DNA damage restricte d to discrete sub n u cle a r regions of the cells. Immunoflourescent staining of MRE11 was found to correlate with the irradiated regions of the nucleus as early as 30 minutes following irradiation, therefore strengthening the argument that irradiation-induced MRE11 foci correspond to sites of DSB repair.

Further evidence that the M/R/N proteins act in DSB repair has come from the phenotypes of cells in which the complex has been disrupted. Null mutations of MRE11, RAD50 or NBS1 result either in cell inviability or embryonic lethality, demonstrating the essential role of these gene products in normal cellular processes (Xiao and Weaver, 1997; Luo etal., 1999; Zhu et al., 2001). More informative are non-null mutations which, when located in MRE11 result in an ataxia telangiectasia-like disorder (ATLD), and within NBS1 cause Nijmegen breakage syndrome (NBS; Carney etal., 1998; Varon etal., 1998; Stewart etal.,

1999). NBS and ATLD patients display many phenotypes seen in ataxia telangiectasia (AT) patients, who carry mutations in the ATM gene, and those with mutations in LIG4 (see Table 1.2, page 57). At the cellular level, deficiency

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in NBS1, MRE11 or A T M results in chromosomal instability and an increased sensitivity to ionising radiation, which indicates the gene products probably act in DNA repair (Shiloh, 1997; Featherstone and Jackson, 1998; Stewart et al.,

1999). Additionally, the cells exhibit radioresistant DNA synthesis brought about by defective G /S checkpoint control, which suggests a supplementary role in the signalling of DNA damage. More recently ATM and the M/R/N complex have been shown to act the same pathway (Wu etal., 2000; Zhao etal., 2000). NBS1 is a substrate for ATM kinase activity, and mutation of the phosphorylation sites resulted in a failure to induce radiation-induced foci and restore radiation resistance to NBS cells. This has confirmed the role of the M/R/N complex in the signalling of DNA damage.

V. Development of cell-free systems to permit

in vitro

analysis of NHEJ