ruvA Mutants that resolve Holliday junctions but do not reverse replication forks.
ruvA Mutants that resolve Holliday junctions but do not reverse replication forks.
复制标题
DOI：
10.1371/journal.pgen.1000012
复制
发表时间：
2008-03-07
期刊：
影响因子：
4.5
通讯作者：
Michel B
中科院分区：
文献类型：
--
作者：
Baharoglu Z;Bradley AS;Le Masson M;Tsaneva I;Michel B
RuvAB和RuvABC复合物分别催化霍利迪连接（HJs）的分支迁移和分解。除了它们在同源重组的最后步骤中的作用外，它们还处理由复制叉逆转产生的HJ，这是一种通过阻断的前导和滞后链末端的退火而在失活的复制叉处发生的反应。RuvAB最近被提议结合复制叉并直接催化它们转化为HJ。我们在这里报告的分离和表征的两个分离的功能ruvA突变体，解决HJ，根据其能力，以促进共轭重组和重组修复的UV和丝裂霉素C病变，但已经失去了能力，以扭转叉。体内和体外证据表明，ruvA突变影响DNA结合和RuvB解旋酶活性的刺激。这项工作表明，RuvA的行动，在叉和在HJ的基因可以分开，和RuvA突变体妥协叉逆转仍然完全能够同源重组。DNA复制是DNA链被复制以确保遗传物质传递到子细胞的过程。染色体复制不是一个连续的过程，而是由于遇到障碍或复制蛋白质的功能障碍而受到意外的抑制。在细菌中，失活的复制叉会重新启动，但它们在重新启动之前通常会被重塑。有趣的是，参与同源重组的酶，重组染色体的过程，也参与叉重塑反应。本研究的主题是RuvAB，一种高度保守的细菌复合物，用作重组中间体解析的模型酶，我们发现它也作用于阻断的分叉。我们在这里描述的分离和表征的ruvA突变体，已专门失去了在失活的复制叉的能力，虽然他们仍然完全能够同源重组。这种ruvA突变体的存在，其性质和纯化的RuvA突变蛋白的性质，表明RuvAB在复制叉的作用比其在重组中间体的作用更苛刻，但在进化过程中仍然被保留。
RuvAB and RuvABC complexes catalyze branch migration and resolution of Holliday junctions (HJs) respectively. In addition to their action in the last steps of homologous recombination, they process HJs made by replication fork reversal, a reaction which occurs at inactivated replication forks by the annealing of blocked leading and lagging strand ends. RuvAB was recently proposed to bind replication forks and directly catalyze their conversion into HJs. We report here the isolation and characterization of two separation-of-function ruvA mutants that resolve HJs, based on their capacity to promote conjugational recombination and recombinational repair of UV and mitomycin C lesions, but have lost the capacity to reverse forks. In vivo and in vitro evidence indicate that the ruvA mutations affect DNA binding and the stimulation of RuvB helicase activity. This work shows that RuvA's actions at forks and at HJs can be genetically separated, and that RuvA mutants compromised for fork reversal remain fully capable of homologous recombination. DNA replication is the process by which DNA strands are copied to ensure the transmission of the genetic material to daughter cells. Chromosome replication is not a continuous process but is subjected to accidental arrests, owing to the encounter of obstacles or to the dysfunctioning of a replication protein. In bacteria, inactivated replication forks restart but they are most often remodeled before restarting. Interestingly, enzymes involved in homologous recombination, the process that rearranges chromosomes, are also involved in fork-remodeling reactions. The subject of the present study is RuvAB, a highly conserved bacterial complex used as the model enzyme for resolution of recombination intermediates, which we found to also act at blocked forks. We describe here the isolation and characterization of ruvA mutants that have specifically lost the capability to act at inactivated replication forks, although they remain fully capable of homologous recombination. The existence of such ruvA mutants, their properties and those of the purified RuvA mutant proteins, indicate that the action of RuvAB at replication forks is more demanding that its action at recombination intermediates, but have nevertheless been preserved during evolution.