Structural basis for DNA 5´-end resection by RecJ.
Structural basis for DNA 5´-end resection by RecJ.
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作者：
Cheng K;Xu H;Chen X;Wang L;Tian B;Zhao Y;Hua Y
在双链断裂时切除5‘端DNA链是重组DNA修复的关键步骤。RECJ是DHH家族蛋白的一员，是参与RECF重组途径的唯一5‘核酸酶。在这里，我们报道了耐辐射变形球菌RecJ与脱氧胸苷单磷酸(DTMP)、单链DNA结合蛋白(SSB-Ct)C-末端区域的络合物的晶体结构，并对RecF途径进行了机制研究。活性部位上方的5‘-磷酸末端结合口袋决定了RecJ脱氧核酸酶的5’-3“极性；活性部位入口处的螺旋通道只接纳单链DNA；蛋白质与9个核苷酸之间的连续堆积作用确保了连续的末端切除。在N-末端结构域中，RecJ的活性中心含有两个与亲核水配位的二价阳离子。单链DNA在具有剪切性的磷酸盐上发生180°转折。RecJ的C-末端结构域与SSB-Ct结合，这解释了RecJ和SSB如何协同工作，有效地处理DNA断端进行同源重组。DOI：http://dx.doi.org/10.7554/eLife.14294.001 DNA编码细胞产生生命所必需的分子和蛋白质所需的信息。因此，迅速准确地修复受损的DNA是至关重要的。一些DNA损伤剂，如伽马辐射，会破坏DNA双螺旋的两条链，如果不快速准确地修复，可能会对细胞造成致命的影响。负责修复这种双链断裂的一个重要途径被称为同源重组修复途径。这种修复的第一阶段包括在断裂处切断其中一条DNA链的一部分。这暴露了一段单链的伙伴链，可用于修复。一种高度抵抗辐射破坏其DNA的生物体是耐辐射球菌。在这种细菌中，一种名为RecJ的酶通过逐渐缩短DNA链的一端来执行修复DNA双链断裂的第一步的一部分。程等人现在已经使用结晶学来观察RecJ与DNA结合时形成的结构。再加上生化实验的结果，揭示了RecJ如何识别在断裂的DNA链上结合的位置，以及它如何在切断DNA链的同时沿着断裂的链移动。进一步的研究发现，另外两种蛋白质增强了RecJ处理断裂DNA链末端的能力。程等人还研究了RecJ与这些额外的蛋白质之一形成的结构，称为SSB。未来的目标是确定这三种蛋白质如何相互协调，以有效和准确地修复耐辐射葡萄球菌细菌的双链断裂。DOI：http://dx.doi.org/10.7554/eLife.14294.002
The resection of DNA strand with a 5´ end at double-strand breaks is an essential step in recombinational DNA repair. RecJ, a member of DHH family proteins, is the only 5´ nuclease involved in the RecF recombination pathway. Here, we report the crystal structures of Deinococcus radiodurans RecJ in complex with deoxythymidine monophosphate (dTMP), ssDNA, the C-terminal region of single-stranded DNA-binding protein (SSB-Ct) and a mechanistic insight into the RecF pathway. A terminal 5´-phosphate-binding pocket above the active site determines the 5´-3´ polarity of the deoxy-exonuclease of RecJ; a helical gateway at the entrance to the active site admits ssDNA only; and the continuous stacking interactions between protein and nine nucleotides ensure the processive end resection. The active site of RecJ in the N-terminal domain contains two divalent cations that coordinate the nucleophilic water. The ssDNA makes a 180° turn at the scissile phosphate. The C-terminal domain of RecJ binds the SSB-Ct, which explains how RecJ and SSB work together to efficiently process broken DNA ends for homologous recombination. DOI: http://dx.doi.org/10.7554/eLife.14294.001 DNA encodes information that cells need to create the molecules and proteins that are essential for life. It is therefore vital that damaged DNA is repaired rapidly and accurately. Some DNA-damaging agents, such as gamma radiation, break both strands of the DNA double helix, which can be fatal to cells if not repaired quickly and accurately. One important pathway in charge of repairing such double-strand breaks is called the homologous recombination repair pathway. The first stage of this repair involves cutting away part of one of the DNA strands at the break. This exposes a single-stranded stretch of the partner strand, which can be used for the repair. One organism that is highly resistant to having its DNA damaged by radiation is the bacterium Deinococcus radiodurans. In this bacterium, an enzyme called RecJ performs part of the first step in the repair of DNA double-strand breaks by progressively shortening one end of a DNA strand. Cheng et al. have now used crystallography to look at the structure that RecJ forms when it binds to DNA. This, together with the results from biochemical experiments, revealed how RecJ recognizes where to bind on a broken DNA strand and how it moves along the broken strand along with cutting that strand. Further investigations revealed that two other proteins enhance the ability of RecJ to process the ends of broken DNA strands. Cheng et al. also examined the structure that RecJ forms with one of these additional proteins, called SSB. A future goal is to determine how all three proteins co-ordinate with each other to efficiently and accurately repair double stranded breaks in the D. radiodurans bacteria. DOI: http://dx.doi.org/10.7554/eLife.14294.002