Doan P L, Belanger K G, Kreuzer K N
Department of Microbiology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Genetics. 2001 Mar;157(3):1077-87. doi: 10.1093/genetics/157.3.1077.
Recombination hotspots have previously been discovered in bacteriophage T4 by two different approaches, marker rescue recombination from heavily damaged phage genomes and recombination during co-infection by two undamaged phage genomes. The phage replication origin ori(34) is located in a region that has a hotspot in both assays. To determine the relationship between the origin and the two kinds of hotspots, we generated phage carrying point mutations that should inactivate ori(34) but not affect the gene 34 reading frame (within which ori(34) is located). The mutations eliminated the function of the origin, as judged by both autonomous replication of plasmids during T4 infection and two-dimensional gel analysis of phage genomic replication intermediates. As expected from past studies, the ori(34) mutations also eliminated the hotspot for marker rescue recombination from UV-irradiated genomes. However, the origin mutations had no effect on the recombination hotspot that is observed with co-infecting undamaged phage genomes, demonstrating that some DNA sequence other than the origin is responsible for inflated recombination between undamaged genomes. The hotspots for marker rescue recombination may result from a replication fork restart process that acts upon origin-initiated replication forks that become blocked at nearby DNA damage. The two-dimensional gel analysis also revealed phage T4 replication intermediates not previously detected by this method, including origin theta forms.
重组热点先前已通过两种不同方法在噬菌体T4中被发现，一种是从严重受损的噬菌体基因组中进行标记拯救重组，另一种是在两个未受损的噬菌体基因组共感染期间进行重组。噬菌体复制起点ori(34)位于在这两种检测中都有热点的区域。为了确定起点与这两种热点之间的关系，我们构建了携带点突变的噬菌体，这些突变应该会使ori(34)失活，但不会影响基因34的阅读框（ori(34)位于该阅读框内）。根据T4感染期间质粒的自主复制以及噬菌体基因组复制中间体的二维凝胶分析判断，这些突变消除了起点的功能。正如过去研究所预期的那样，ori(34)突变也消除了来自紫外线照射基因组的标记拯救重组热点。然而，起点突变对未受损噬菌体基因组共感染时观察到的重组热点没有影响，这表明除起点外的一些DNA序列是未受损基因组之间重组增强的原因。标记拯救重组热点可能源于复制叉重新启动过程，该过程作用于在附近DNA损伤处受阻的由起点引发的复制叉。二维凝胶分析还揭示了噬菌体T4复制中间体，这些中间体以前未通过这种方法检测到，包括起点θ型。
