Báo cáo khoa học: Analysis of DNA-binding sites on Mhr1, a yeast mitochondrial ATP-independent

Analysis of DNA-binding sites on Mhr1, a yeast

mitochondrial ATP-independent homologous pairing

protein

Tokiha Masuda1,2, Feng Ling2

, Takehiko Shibata1,2 and Tsutomu Mikawa1,2,3

1 Graduate School of Nanobioscience, Yokohama City University, Japan

2 RIKEN Advanced Science Institute, Saitama, Japan

3 RIKEN SPring-8 Center, Hyogo, Japan

Introduction

Homologous DNA recombination is conserved in all

organisms. In the nucleus, homologous recombination

is involved in the maintenance of genome integrity

during mitosis, and in genetic diversification through

meiosis. In bacteria, homologous recombination

strictly depends on the RecA gene [1–4], whereas in

eukaryotes it depends on the Rad51 [5–8] and Dmc1

[9–12] genes, both of which encode RecA orthologs.

Homologous recombination is initiated via a single￾stranded gap or a double-strand break, which is

processed to produce 3¢-ssDNA tails [13]. Each ssDNA

region invades undamaged homologous dsDNA,

resulting in the formation of homologous joints

between the dsDNA and ssDNA through the pairing

of complementary sequences. This reaction is termed

homologous pairing (HP), and it is followed by a

strand exchange to stabilize the joint [1,14]. The

RecA ⁄Rad51 family of proteins promotes HP, which is

a key process of homologous recombination, in an

ATP-dependent manner in vitro.

Keywords

fluorescence resonance energy transfer

(FRET); homologous recombination; Mhr1;

mtDNA; RecA

Correspondence

T. Mikawa, RIKEN Advanced Science

Institute, 1-7-29 Suehiro-cho, Tsurumi-ku,

Yokohama 230-0045, Japan

Fax: +81 45 5087364

Tel: +81 45 5087224

E-mail: [email protected]

(Received 6 October 2009, revised 24

December 2009, accepted 8 January 2010)

doi:10.1111/j.1742-4658.2010.07574.x

The Mhr1 protein is necessary for mtDNA homologous recombination in

Saccharomyces cerevisiae. Homologous pairing (HP) is an essential reaction

during homologous recombination, and is generally catalyzed by the

RecA ⁄Rad51 family of proteins in an ATP-dependent manner. Mhr1 cata￾lyzes HP through a mechanism similar, at the DNA level, to that of the

RecA ⁄Rad51 proteins, but without utilizing ATP. However, it has no

sequence homology with the RecA⁄Rad51 family proteins or with other

ATP-independent HP proteins, and exhibits different requirements for

DNA topology. We are interested in the structural features of the func￾tional domains of Mhr1. In this study, we employed the native fluorescence

of Mhr1’s Trp residues to examine the energy transfer from the Trp resi￾dues to etheno-modified ssDNA bound to Mhr1. Our results showed that

two of the seven Trp residues (Trp71 and Trp165) are spatially close to the

bound DNA. A systematic analysis of mutant Mhr1 proteins revealed that

Asp69 is involved in Mg2+-dependent DNA binding, and that multiple Lys

and Arg residues located around Trp71 and Trp165 are involved in the

DNA-binding activity of Mhr1. In addition, in vivo complementation anal￾yses showed that a region around Trp165 is important for the maintenance

of mtDNA. On the basis of these results, we discuss the function of the

region surrounding Trp165.

Abbreviations

dnRad54, Danio rerio Rad54; FRET, fluorescence resonance energy transfer; HP, homologous pairing; essDNA, etheno-ssDNA.

1440 FEBS Journal 277 (2010) 1440–1452 ª 2010 The Authors Journal compilation ª 2010 FEBS