Tài liệu Báo cáo khoa học: DNA strand exchange activity of rice recombinase OsDmc1 monitored by

DNA strand exchange activity of rice recombinase

OsDmc1 monitored by fluorescence resonance energy

transfer and the role of ATP hydrolysis

Chittela Rajanikant1

, Manoj Kumbhakar2

, Haridas Pal2

, Basuthkar J. Rao3 and Jayashree K. Sainis1

1 Molecular Biology Division, Bhabha Atomic Research Center, Mumbai, India

2 Radiation Chemistry and Chemical Dynamics Division, Bhabha Atomic Research Center, Mumbai, India

3 Department of Biological Sciences, Tata Institute of Fundamental Research, Mumbai, India

Homologous recombination is a fundamental process

by which two DNA molecules physically interact with

each other. This process is important for repairing the

double strand breaks (DSBs) induced during mitosis,

meiosis and other stages where chromosomal break￾ages ensue. There are several sequential biochemical

Keywords

Dmc1; FRET; renaturation; rice; strand

exchange

Correspondence

J. K. Sainis, Molecular Biology Division,

Bhabha Atomic Research Centre,

Mumbai 400 085, India

Fax: +91 22 25505326

Tel : +91 22 25595079

E-mail: [email protected]

(Received 18 October 2005, revised 2

February 2006, accepted 8 February 2006)

doi:10.1111/j.1742-4658.2006.05170.x

Rad51 and disrupted meiotic cDNA1 (Dmc1) are the two eukaryotic DNA

recombinases that participate in homology search and strand exchange

reactions during homologous recombination mediated DNA repair. Rad51

expresses in both mitotic and meiotic tissues whereas Dmc1 is confined to

meiosis. DNA binding and pairing activities of Oryza sativa disrupted mei￾otic cDNA1 (OsDmc1) from rice have been reported earlier. In the present

study, DNA renaturation and strand exchange activities of OsDmc1 have

been studied, in real time and without the steps of deproteinization, using

fluorescence resonance energy transfer (FRET). The extent as well as the

rate of renaturation is the highest in conditions that contain ATP, but sig￾nificantly less when ATP is replaced by slowly hydrolysable analogues of

ATP, namely adenosine 5¢-(b,c-imido) triphosphate (AMP-PNP) or adeno￾sine 5¢-O-(3-thio triphosphate) (ATP-c-S), where the former was substan￾tially poorer than the latter in facilitating the renaturation function. FRET

assay results also revealed OsDmc1 protein concentration dependent strand

exchange function, where the activity was the fastest in the presence of

ATP, whereas in the absence of a nucleotide cofactor it was several fold

(
15-fold) slower. Interestingly, strand exchange, in reactions where ATP

was replaced with AMP-PNP or ATP-c-S, was somewhat slower than that

of even minus nucleotide cofactor control. Notwithstanding the slow rates,

the reactions with no nucleotide cofactor or with ATP-analogues did reach

the same steady state level as seen in ATP reaction. FRET changes were

unaffected by the steps of deproteinization following OsDmc1 reaction,

suggesting that the assay results reflected stable events involving exchanges

of homologous DNA strands. All these results, put together, suggest that

OsDmc1 catalyses homologous renaturation as well as strand exchange

events where ATP hydrolysis seems to critically decide the rates of the reac￾tion system. These studies open up new facets of a plant recombinase func￾tion in relation to the role of ATP hydrolysis.

Abbreviations

AMP-PNP, adenosine 5¢-(b,c-imido) triphosphate; ATP-c-S, adenosine 5¢-O-(3-thio triphosphate); Dmc1, disrupted meiotic cDNA1; DS, double

stranded; DSBs, double strand breaks; FRET, fluorescence resonance energy transfer; OsDmc1, Oryza sativa disrupted meiotic cDNA1;

SS, single stranded; RecA, DNA recombinase A; RPA, replication protein A.

FEBS Journal 273 (2006) 1497–1506 ª 2006 The Authors Journal compilation ª 2006 FEBS 1497