Shcherbakov Victor P, Plugina Lidiya, Shcherbakova Tamara, Sizova Svetlana, Kudryashova Elena
Institute of Problems of Chemical Physics RAS, Chernogolovka, Moscow Region 142432, Russia.
DNA Repair (Amst). 2006 Jul 13;5(7):773-87. doi: 10.1016/j.dnarep.2006.03.007. Epub 2006 May 22.
Coordination of DNA ends during double-strand break (DSB) repair was studied in crosses of bacteriophage T4 in which DSBs were induced site-specifically by SegC endonuclease in the DNA of only one of the parents. Coupling of the genetic exchanges to the left and to the right of the DSB was measured in the wild-type genetic background as well as in T4 strains bearing mutations in several recombination genes: 47, uvsX, uvsW, 59, 39 and 61. The observed quantitative correlation between the degree of coupling and position of the recombining markers in relation to the DSB point implies that the two variants of the splice/patch-coupling (SPC) pathway, the "sequential SPC" and the "SPC with fork collision", operate during DSB repair. In the 47 mutant with or without a das suppressor, coupling of the exchanges was greatly reduced, indicating a crucial role of the 47/46 complex in coupling of the genetic exchanges on the two sides of the DSB. From the observed dependence of the apparent coupling on the intracellular ratio of breakable and unbreakable chromosomes in different genetic backgrounds it is inferred that linking of the DNA ends by 47/46 protein is the mechanism that accounts for their concerted action during DSB repair. A mechanism of replicative resolution of D-loop intermediate (RR pathway) is suggested to explain the phenomenology of DSB repair in DNA arrest and uvsW mutants. A "left"-"right" bias in the recombinogenic action of two DNA ends of the broken chromosome was observed which was particularly prominent in the 59 (41-helicase loader) and 39 (topoisomerase) mutants. Phage topoisomerase II (gp39-52-60) is indispensable for growth in the DNA arrest mutants: the doubles 47(-)39(-), uvsX 39(-) and 59(-)39(-) are lethal.
在噬菌体T4的杂交实验中研究了双链断裂（DSB）修复过程中DNA末端的协调作用，其中双链断裂由SegC核酸内切酶在仅一方亲本的DNA中进行位点特异性诱导。在野生型遗传背景以及携带几个重组基因突变（47、uvsX、uvsW、59、39和61）的T4菌株中，测量了DSB左侧和右侧遗传交换的耦合情况。观察到的耦合程度与重组标记相对于DSB点的位置之间的定量相关性表明，剪接/补丁耦合（SPC）途径的两种变体，即“顺序SPC”和“叉碰撞SPC”，在DSB修复过程中起作用。在有或没有das抑制子的47突变体中，交换的耦合大大降低，表明47/46复合物在DSB两侧遗传交换的耦合中起关键作用。从在不同遗传背景下观察到的表观耦合对可断裂和不可断裂染色体内细胞比例的依赖性可以推断，47/46蛋白连接DNA末端是其在DSB修复过程中协同作用的机制。提出了一种D环中间体的复制性解离机制（RR途径）来解释DNA停滞和uvsW突变体中DSB修复的现象学。观察到断裂染色体的两个DNA末端的重组作用存在“左”-“右”偏向，这在59（41-解旋酶装载蛋白）和39（拓扑异构酶）突变体中尤为明显。噬菌体拓扑异构酶II（gp39-52-60）对于DNA停滞突变体的生长是不可或缺的：双突变体47（-）39（-）、uvsX 39（-）和59（-）39（-）是致死的。
