Mechanism of DNA excision repair based on the three-dimensional structure of UvrABC endonuclease

RecJ是一种单链DNA的5‘-3’外切酶，参与DNA修复和重组系统。最近，RecJ被认为是复制-重组关系的关键酶。RecJ同源物有5个特征基序，组成了一个预测的磷酸酯酶大家族，命名为DHH家族。我们对Thermus Therhilus HB8的RecJ(TtRecJ)进行了X射线结晶学研究，将其表达为包涵体，通过变性和复性得到纯化。限制性蛋白水解法鉴定催化核心区(CD-ttRecJ)。以可溶性形式表达的Cd-ttRecJ被高度提纯和结晶，采用单波长反常分散的方法解决了Cd-ttRecJ的结构问题。RecJ有一个新颖的折叠，其中两个结构域通过一个长螺旋相互连接，形成一个中心凹槽。该沟由保守的残基和带正电荷的残基组成，可能参与DNA结合。金属离子通过DHH家族特有的基序配位。通过反常散射实验，确定该金属离子为Mn^&lt；2&gt；

RecJ is a 5' to 3' exonuclease specific for single-stranded DNA, and involved in DNA repair and recombination systems. Recently, RecJ has been highlighted as a key enzyme for the replication-recombination relationship. RecJ homologues have five characteristic motifs, which form a large family of the predicted phosphoesterases, named by DHH family.We performed the X-ray crystallography of RecJ from Thermus thermohilus HB8 (ttRecJ).As ttRecJ was expressed as an inclusion body, it was purified through denaturation and refolding. The region of the catalytic core domain (cd-ttRecJ) was identified by limited proteolysis. Cd-ttRecJ, expressed as soluble form, was highly purified and crystallized.The structure of RecJ was solved by single-wavelength anomalous dispersion, using Se-Met cd-ttRecJ. RecJ has a novel fold, in which two domains are interconnected with a long helix to form a central groove. This groove is composed of conserved residues and positively charged, which may be involved in DNA binding. The metal ion is coordinated by the motifs characteristic in DHH family. This metal ion was identified as Mn^<2+> by an anomalous scattering experiment.