Culyba Matthew J, Minkah Nana, Hwang Young, Benhamou Ori-Michael J, Bushman Frederic D
Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6076, USA.
J Biol Chem. 2007 Nov 30;282(48):34644-52. doi: 10.1074/jbc.M705322200. Epub 2007 Sep 21.
DNA replication, recombination, and repair can result in formation of diverse branched DNA structures. Many large DNA viruses are known to encode DNA branch nucleases, but several of the expected activities have not previously been found among poxvirus enzymes. Vaccinia encodes an enzyme, A22 resolvase, which is known to be active on four-stranded DNA junctions (Holliday junctions) or Holliday junction-like structures containing three of the four strands. Here we report that A22 resolvase in fact has a much wider substrate specificity than previously appreciated. A22 resolvase cleaves Y-junctions, single-stranded DNA flaps, transitions from double strands to unpaired single strands ("splayed duplexes"), and DNA bulges in vitro. We also report site-directed mutagenesis studies of candidate active site residues. The results identify the likely active site and support a model in which a single active site is responsible for cleavage on Holliday junctions and splayed duplexes. Lastly, we describe possible roles for the A22 resolvase DNA-branch nuclease activity in DNA replication and repair.
DNA复制、重组和修复可导致形成多种分支DNA结构。已知许多大型DNA病毒编码DNA分支核酸酶，但痘病毒酶中尚未发现一些预期的活性。痘苗病毒编码一种酶，即A22解离酶，已知其对四链DNA连接点（霍利迪连接点）或含有四条链中三条链的类霍利迪连接点结构具有活性。在此我们报告，实际上A22解离酶的底物特异性比之前所认识的要广泛得多。A22解离酶在体外可切割Y型连接点、单链DNA瓣、双链到未配对单链的转变（“展开双链体”）以及DNA凸起。我们还报告了对候选活性位点残基的定点诱变研究。结果确定了可能的活性位点，并支持一个模型，即单个活性位点负责霍利迪连接点和展开双链体的切割。最后，我们描述了A22解离酶DNA分支核酸酶活性在DNA复制和修复中的可能作用。
