Structural and functional characterization of the Redβ recombinase from bacteriophage λ.
Structural and functional characterization of the Redβ recombinase from bacteriophage λ.
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DOI：
10.1371/journal.pone.0078869
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Heddle JG
中科院分区：
文献类型：
--
作者：
Matsubara K;Malay AD;Curtis FA;Sharples GJ;Heddle JG
噬菌体λ的红色系统负责基因重排，这有助于其快速进化，并已成功地用作基因组操纵的研究工具。关键的重组组分是Redβ，一种促进互补DNA链退火的环状蛋白。Redβ与人类Rad 52单链DNA（ssDNA）退火蛋白具有功能相似性，尽管它们的进化相关性尚未完全确定。Rad 52和Redβ序列的比对显示出总体低水平的同源性，在N-末端核心结构域中具有15%的同一性以及与来自噬菌体ul 36的Rad 52同源物Sak的重要相似性。选择关键的保守残基进行诱变，并探测它们对寡聚体形成、ssDNA结合和退火的影响。两个保守的区域被确定为重要的结合ssDNA的网站，一个表面的基本集群和亚基间疏水补丁，与Rad 52的研究结果一致。令人惊讶的是，Rad 52中涉及ssDNA结合的碱性簇中的Redβ残基的突变破坏了寡聚体形成和ssDNA结合。Rad 52亚基间疏水斑块的突变不影响Redβ寡聚化，但会损害DNA结合和退火。我们还鉴定了一个对寡聚化和DNA结合几乎没有影响但抑制DNA退火的单个氨基酸取代，表明Redβ的这两种功能可以分开。总之，这些结果为Redβ功能的结构基础和四级结构的重要作用提供了新的见解。
The Red system of bacteriophage λ is responsible for the genetic rearrangements that contribute to its rapid evolution and has been successfully harnessed as a research tool for genome manipulation. The key recombination component is Redβ, a ring-shaped protein that facilitates annealing of complementary DNA strands. Redβ shares functional similarities with the human Rad52 single-stranded DNA (ssDNA) annealing protein although their evolutionary relatedness is not well established. Alignment of Rad52 and Redβ sequences shows an overall low level of homology, with 15% identity in the N-terminal core domains as well as important similarities with the Rad52 homolog Sak from phage ul36. Key conserved residues were chosen for mutagenesis and their impact on oligomer formation, ssDNA binding and annealing was probed. Two conserved regions were identified as sites important for binding ssDNA; a surface basic cluster and an intersubunit hydrophobic patch, consistent with findings for Rad52. Surprisingly, mutation of Redβ residues in the basic cluster that in Rad52 are involved in ssDNA binding disrupted both oligomer formation and ssDNA binding. Mutations in the equivalent of the intersubunit hydrophobic patch in Rad52 did not affect Redβ oligomerization but did impair DNA binding and annealing. We also identified a single amino acid substitution which had little effect on oligomerization and DNA binding but which inhibited DNA annealing, indicating that these two functions of Redβ can be separated. Taken together, the results provide fresh insights into the structural basis for Redβ function and the important role of quaternary structure.
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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：
3.2
作者：
MURPHY, KC
通讯作者：
MURPHY, KC
影响因子：
5.6
作者：
Karakousis, G;Ye, N;Radding, CM
通讯作者：
Radding, CM
影响因子：
3.5
作者：
Mythili, E;Kumar, KA;Muniyappa, K
通讯作者：
Muniyappa, K
影响因子：
14.9
作者：
Krejci L;Altmannova V;Spirek M;Zhao X
通讯作者：
Zhao X