Rajanikant Chittela, Kumbhakar Manoj, Pal Haridas, Rao Basuthkar J, Sainis Jayashree K
Molecular Biology Division, Bhabha Atomic Research Center, Mumbai, India.
FEBS J. 2006 Apr;273(7):1497-506. 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 meiotic 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 significantly less when ATP is replaced by slowly hydrolysable analogues of ATP, namely adenosine 5'-(beta,gamma-imido) triphosphate (AMP-PNP) or adenosine 5'-O-(3-thio triphosphate) (ATP-gamma-S), where the former was substantially 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 ( approximately 15-fold) slower. Interestingly, strand exchange, in reactions where ATP was replaced with AMP-PNP or ATP-gamma-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 reaction system. These studies open up new facets of a plant recombinase function in relation to the role of ATP hydrolysis.
Rad51和减数分裂破坏cDNA1（Dmc1）是两种真核生物DNA重组酶，它们在同源重组介导的DNA修复过程中参与同源性搜索和链交换反应。Rad51在有丝分裂和减数分裂组织中均有表达，而Dmc1仅局限于减数分裂过程。此前已有报道来自水稻的水稻减数分裂破坏cDNA1（OsDmc1）的DNA结合和配对活性。在本研究中，利用荧光共振能量转移（FRET）实时且无需脱蛋白步骤，对OsDmc1的DNA复性和链交换活性进行了研究。在含有ATP的条件下，复性程度和速率最高，但当ATP被ATP的缓慢水解类似物，即腺苷5'-（β，γ-亚氨基）三磷酸（AMP-PNP）或腺苷5'-O-（3-硫代三磷酸）（ATP-γ-S）取代时，复性程度和速率显著降低，其中前者在促进复性功能方面明显不如后者。FRET分析结果还揭示了OsDmc1蛋白浓度依赖性的链交换功能，在有ATP存在时活性最快，而在没有核苷酸辅因子时则慢几倍（约15倍）。有趣的是，在ATP被AMP-PNP或ATP-γ-S取代的反应中，链交换甚至比无核苷酸辅因子对照反应还要慢一些。尽管速率较慢，但无核苷酸辅因子或ATP类似物的反应确实达到了与ATP反应相同的稳态水平。OsDmc1反应后的脱蛋白步骤不影响FRET变化，这表明分析结果反映了涉及同源DNA链交换的稳定事件。综合所有这些结果表明，OsDmc1催化同源复性以及链交换事件，其中ATP水解似乎对反应系统的速率起着关键决定作用。这些研究揭示了植物重组酶功能与ATP水解作用相关的新方面。
