Mutational Analysis of Redβ Single Strand Annealing Protein: Roles of the 14 Lysine Residues in DNA Binding and Recombination In Vivo.
Mutational Analysis of Redβ Single Strand Annealing Protein: Roles of the 14 Lysine Residues in DNA Binding and Recombination In Vivo.
复制标题
Redβ 单链退火蛋白的突变分析：14 个赖氨酸残基在体内 DNA 结合和重组中的作用。
DOI：
10.3390/ijms22147758
复制
发表时间：
2021-07-20
影响因子：
5.6
通讯作者：
Bell CE
中科院分区：
文献类型：
--
作者：
Zakharova K;Caldwell BJ;Ta S;Wheat CT;Bell CE
Redβ是来自λ噬菌体的261个氨基酸的蛋白质，其促进单链退火（SSA）反应以修复双链DNA（dsDNA）断裂。虽然目前还没有Redβ的高分辨率结构，但基于与人Rad 52的同源性及其C-末端结构域（CTD，残基193-261）的晶体结构，提出了其DNA结合结构域（残基1-188）的模型，该结构域结合λ核酸外切酶和E。大肠杆菌单链DNA结合蛋白（SSB）。为了评估这些模型，将Redβ的14个赖氨酸残基突变为丙氨酸，并测试变体的体内重组和体外DNA结合和退火。DNA结合结构域中的大多数赖氨酸，包括K36、K61、K111、K132、K148、K154和K172，被发现对于体外DNA结合和体内重组是关键的。相比之下，CTD内的赖氨酸，包括K214、K245、K251、K253和K258，都不是体外DNA结合所需的，但是K214和K253这两个赖氨酸对于体内重组是关键的，这可能是由于它们参与了与SSB的结合。K61被鉴定为对DNA退火至关重要的残基，但不是初始ssDNA结合的残基，这表明K61在结合结合到复合物中的DNA的第二链中起作用。K148 A变异体，先前已被证明在寡聚体形成中有缺陷，对ssDNA的亲和力最低，并且是唯一完全不合作的变异体，表明ssDNA结合与寡聚化偶联。
Redβ is a 261 amino acid protein from bacteriophage λ that promotes a single-strand annealing (SSA) reaction for repair of double-stranded DNA (dsDNA) breaks. While there is currently no high-resolution structure available for Redβ, models of its DNA binding domain (residues 1–188) have been proposed based on homology with human Rad52, and a crystal structure of its C-terminal domain (CTD, residues 193-261), which binds to λ exonuclease and E. coli single-stranded DNA binding protein (SSB), has been determined. To evaluate these models, the 14 lysine residues of Redβ were mutated to alanine, and the variants tested for recombination in vivo and DNA binding and annealing in vitro. Most of the lysines within the DNA binding domain, including K36, K61, K111, K132, K148, K154, and K172, were found to be critical for DNA binding in vitro and recombination in vivo. By contrast, none of the lysines within the CTD, including K214, K245, K251, K253, and K258 were required for DNA binding in vitro, but two, K214 and K253, were critical for recombination in vivo, likely due to their involvement in binding to SSB. K61 was identified as a residue that is critical for DNA annealing, but not for initial ssDNA binding, suggesting a role in binding to the second strand of DNA incorporated into the complex. The K148A variant, which has previously been shown to be defective in oligomer formation, had the lowest affinity for ssDNA, and was the only variant that was completely non-cooperative, suggesting that ssDNA binding is coupled to oligomerization.
登录
查看更多内容
影响因子：
3.7
作者：
Matsubara K;Malay AD;Curtis FA;Sharples GJ;Heddle JG
通讯作者：
Heddle JG
DOI：
10.1073/pnas.121164898
发表时间：
2001-06-05
影响因子：
11.1
作者：
Ellis, HM;Yu, DG;Court, DL
通讯作者：
Court, DL
影响因子：
14.9
作者：
Caldwell, Brian J.;Zakharova, Ekaterina;Bell, Charles E.
通讯作者：
Bell, Charles E.
DOI：
10.1016/j.pbiomolbio.2019.03.005
发表时间：
2019-10
影响因子：
3.8
作者：
Caldwell BJ;Bell CE
通讯作者：
Bell CE
影响因子：
5.6
作者：
Makhov, Alexander M.;Sen, Anindito;Egelman, Edward H.
通讯作者：
Egelman, Edward H.