Robertson Ragan B, Moses Dana N, Kwon YoungHo, Chan Pamela, Chi Peter, Klein Hannah, Sung Patrick, Greene Eric C
Department of Biological Sciences and Biochemistry, Columbia University, 650 West 168th Street, New York, NY 10032, USA.
Proc Natl Acad Sci U S A. 2009 Aug 4;106(31):12688-93. doi: 10.1073/pnas.0811465106. Epub 2009 Jul 21.
Rad51 is a core component of the eukaryotic homologous recombination machinery and is responsible for key mechanistic steps during strand invasion. Higher order oligomers of Rad51 display a remarkable degree of structural variation, forming rings, compressed filaments, and elongated filaments. It is unclear whether Rad51 can transition directly between these different oligomeric structures without disassembling first into monomers. We have used single-molecule microscopy to investigate the behavior of human Rad51 assembled on double-stranded DNA. Our results show that human Rad51 can form elongated nucleoprotein filaments on DNA, but ATP hydrolysis causes a decrease in their length without concomitant dissociation of protein. Compressed Rad51 filaments can re-elongate when presented with either ATP or the non-hydrolyzable analog AMP-PNP, and these cycles of elongation and compression are reversible. A Rad51 mutant deficient in ATP hydrolysis is locked into an extended conformation that is incapable of transitioning to a compressed filament. Similarly, wild-type Rad51 bound to DNA in the presence of AMP-PNP was trapped in the elongated state. Proteins incapable of transitioning to the compressed state were also highly resistant to dissociation from the DNA. Taken together, our results indicate that nucleotide hydrolysis by human Rad51 triggers a reversible structural transition leading to filaments with reduced helical pitch.
Rad51是真核生物同源重组机制的核心组成部分，负责链侵入过程中的关键机制步骤。Rad51的高阶寡聚体表现出显著程度的结构变异，形成环状、压缩丝状和伸长丝状。尚不清楚Rad51是否能在不先解聚成单体的情况下直接在这些不同的寡聚体结构之间转变。我们利用单分子显微镜研究了组装在双链DNA上的人类Rad51的行为。我们的结果表明，人类Rad51能在DNA上形成伸长的核蛋白丝，但ATP水解会导致其长度缩短，而蛋白质不会随之解离。当提供ATP或不可水解的类似物AMP-PNP时，压缩的Rad51丝可以重新伸长，并且这些伸长和压缩循环是可逆的。缺乏ATP水解能力的Rad51突变体被锁定在一种伸展构象中，无法转变为压缩丝状。同样，在AMP-PNP存在下与DNA结合的野生型Rad51被困在伸长状态。无法转变为压缩状态的蛋白质也对从DNA上解离具有高度抗性。综上所述，我们的结果表明，人类Rad51的核苷酸水解触发了一种可逆的结构转变，导致螺旋间距减小的丝。
