Tài liệu Báo cáo khoa học: Roles of the human Rad51 L1 and L2 loops in DNA binding doc

Roles of the human Rad51 L1 and L2 loops in DNA binding

Yusuke Matsuo1

, Isao Sakane2

, Yoshimasa Takizawa1

, Masayuki Takahashi3

and Hitoshi Kurumizaka1,2

1 Graduate School of Science and Engineering, Waseda University, Tokyo, Japan

2 Institute for Biochemical Engineering, Waseda University, Tokyo, Japan

3 UMR 6204 Biocatalyse-Biotechnologie-Bioregulation, Centre National de la Recherche Scientifique, and University of Nantes, France

The Rad51 proteins are the eukaryotic orthologs of

the bacterial RecA protein [1], which promotes key

steps in homologous recombination [2–5]. A RAD51

null mutation causes severe defects in meiotic homol￾ogous recombination and mitotic recombinational

repair of double strand breaks (DSBs) in Saccharomy￾ces cerevisiae [1]. Rad51 is thus required for both the

meiotic and mitotic homologous recombination pro￾cesses, while another ortholog, Dmc1, is specific to

meiotic homologous recombination [6–8]. In higher

eukaryotes, Rad51 is even essential for cell survival:

disruption of the RAD51 gene in mice results in early

embryonic lethality [9,10] and the RAD51 gene knock￾out in chicken DT40 cells causes cell death, with the

accumulation of spontaneous chromosomal breaks

[11].

Rad51 and RecA apparently use similar mechanisms

to promote homologous recombination [12–15]. Dur￾ing the homologous recombination process, Rad51 is

thought to bind single-stranded tails produced at DSB

sites, and to form a helical nucleoprotein filament. The

single-stranded DNA (ssDNA) and double-stranded

Keywords

DNA binding; DNA repair; Rad51; Rad51

mutant; recombination

Correspondence

H. Kurumizaka, Graduate School of Science

and Engineering, Waseda University, 3-4-1

Okubo, Shinjuku-ku, Tokyo 169-8555, Japan

Fax: +81 3 5292 9211

Tel: +81 3 5286 8189

E-mail: [email protected]

(Received 12 November 2005, revised

3 April 2006, accepted 16 May 2006)

doi:10.1111/j.1742-4658.2006.05323.x

The human Rad51 protein, a eukaryotic ortholog of the bacterial RecA

protein, is a key enzyme that functions in homologous recombination and

recombinational repair of double strand breaks. The Rad51 protein con￾tains two flexible loops, L1 and L2, which are proposed to be sites for

DNA binding, based on a structural comparison with RecA. In the present

study, we performed mutational and fluorescent spectroscopic analyses on

the L1 and L2 loops to examine their role in DNA binding. Gel retarda￾tion and DNA-dependent ATP hydrolysis measurements revealed that the

substitution of the tyrosine residue at position 232 (Tyr232) within the L1

loop with alanine, a short side chain amino acid, significantly decreased the

DNA-binding ability of human Rad51, without affecting the protein fold￾ing or the salt-induced, DNA-independent ATP hydrolysis. Even the

conservative replacement with tryptophan affected the DNA binding,

indicating that Tyr232 is involved in DNA binding. The importance of the

L1 loop was confirmed by the fluorescence change of a tryptophan residue,

replacing the Asp231, Ser233, or Gly236 residue, upon DNA binding. The

alanine replacement of phenylalanine at position 279 (Phe279) within the

L2 loop did not affect the DNA-binding ability of human Rad51, unlike

the Phe203 mutation of the RecA L2 loop. The Phe279 side chain may not

be directly involved in the interaction with DNA. However, the fluores￾cence intensity of the tryptophan replacing the Rad51-Phe279 residue was

strongly reduced upon DNA binding, indicating that the L2 loop is also

close to the DNA-binding site.

Abbreviations

DSB, double strand break; dsDNA, double-stranded DNA; HsRad51, Homo sapiens Rad51; RPA, replication protein A; ScRad51,

Saccharomyces cerevisiae Rad51; ssDNA, single-stranded DNA; SSB, single stranded DNA-binding protein.

3148 FEBS Journal 273 (2006) 3148–3159 ª 2006 The Authors Journal compilation ª 2006 FEBS