Maxwell Karen L, Reed Patricia, Zhang Rong-Guang, Beasley Steven, Walmsley Adrian R, Curtis Fiona A, Joachimiak Andrej, Edwards Aled M, Sharples Gary J
Centre for Infectious Diseases, Wolfson Research Institute, University of Durham, Queen's Campus, Stockton-on-Tees TS17 6BH, United Kingdom.
Proc Natl Acad Sci U S A. 2005 Aug 9;102(32):11260-5. doi: 10.1073/pnas.0503399102. Epub 2005 Aug 2.
Genetic recombination in bacteriophage lambda relies on DNA end processing by Exo to expose 3'-tailed strands for annealing and exchange by beta protein. Phage lambda encodes an additional recombinase, Orf, which participates in the early stages of recombination by supplying a function equivalent to the Escherichia coli RecFOR complex. These host enzymes assist loading of the RecA strand exchange protein onto ssDNA coated with ssDNA-binding protein. In this study, we purified the Orf protein, analyzed its biochemical properties, and determined its crystal structure at 2.5 angstroms. The homodimeric Orf protein is arranged as a toroid with a shallow U-shaped cleft, lined with basic residues, running perpendicular to the central cavity. Orf binds DNA, favoring single-stranded over duplex and with no obvious preference for gapped, 3'-tailed, or 5'-tailed substrates. An interaction between Orf and ssDNA-binding protein was indicated by far Western analysis. The functional similarities between Orf and RecFOR are discussed in relation to the early steps of recombinational exchange and the interplay between phage and bacterial recombinases.
噬菌体λ中的基因重组依赖于Exo对DNA末端的加工，以暴露出3'尾链，供β蛋白进行退火和交换。噬菌体λ编码另一种重组酶Orf，它通过提供与大肠杆菌RecFOR复合物等效的功能参与重组的早期阶段。这些宿主酶协助将RecA链交换蛋白加载到包被有单链DNA结合蛋白的单链DNA上。在本研究中，我们纯化了Orf蛋白，分析了其生化特性，并确定了其2.5埃的晶体结构。同源二聚体Orf蛋白排列成一个具有浅U形裂隙的环面，裂隙内衬有碱性残基，垂直于中央腔。Orf结合DNA，相比于双链更倾向于单链，对有缺口的、3'尾或5'尾底物没有明显偏好。远缘Western分析表明Orf与单链DNA结合蛋白之间存在相互作用。结合重组交换的早期步骤以及噬菌体和细菌重组酶之间的相互作用，讨论了Orf与RecFOR之间的功能相似性。
