INITIATION OF DNA REPLICATION IN THE PHAGE T4 SYSTEM

这个项目的长期目标是阐明分子 T4噬菌体启动DNA复制的机制，以及 为了了解噬菌体复制之间的关系， 重组和转录。从惊人的相似之处判断 在T4和真核细胞的DNA代谢机制中，噬菌体 T4模型系统可能与人类研究特别相关 染色体的复制、重组和修复，从而对人类 健康。 两个特征良好的T4复制起始点包含中间模式 启动子和DNA的下游片段，很容易解开。这个 启动子和下游区域在启动中的作用将是 探索，使用原始DNA突变和缺乏 特定的复制蛋白。一个有趣的模型是 启动子和去卷曲区域的组合导致稳定的 解旋区的RNA-DNA杂交体。这款车型将直接 在体外进行了测试，结果可能对 包括转录和DNA复制。一种新的转座子系统 突变也将被用来寻找更多涉及到 起源依赖的复制和T4重组过程中。 依赖重组的T4复制的机制将是 使用一种新的系统进行了探索，其中DNA复制是由 病毒基因组中的定点双链断裂。特定的 将分析依赖重组的复制的中间产物 在野生型和各种突变感染中，以及对 同源DNA片段将进一步探索。 T4重组的直接研究也将利用特定的位点 噬菌体基因组中的双链断裂。单链退火法 长期以来，T4噬菌体一直被认为是基因重组的模型 最近出现了在重复的真核生物之间的重组 基因。这一模型将通过分析DNA的重组来检验 能够提供互补单链区域的分子 与双链断裂相邻。重组的机制是在 还将研究天然的T4重组热点，特别是 因为这些热点是由上述复制触发的 病毒的起源。

The long-term goals of this project are to elucidate the molecular mechanisms used by bacteriophage T4 to initiate DNA replication, and to understand the relationships between phage replication, recombination and transcription. As judged by striking similarities in the DNA metabolic machineries of T4 and eukaryotic cells, the phage T4 model system may be particularly relevant to studies of human chromosomal replication, recombination and repair, and thus to human health. Two well-characterized T4 replication origins contain a middle-mode promoter and a downstream segment of DNA that readily unwinds. The roles of the promoter and downstream region in initiation will be explored, using origin DNA mutations and T4 mutants that lack particular replication proteins. One interesting model is that the combination of a promoter and an unwinding region leads to a stable RNA-DNA hybrid in the unwinding region. This model will be directly tested in vitro, and the results could have important implications for both transcription and DNA replication. A new system of transposon mutagenesis will also be used to find additional genes involved in origin-dependent replication and in T4 recombinational processes. The mechanism of recombination-dependent T4 replication will be explored using a novel system in which DNA replication is triggered by site-specific double-strand breaks in the viral genome. Specific intermediates in recombination-dependent replication will be analyzed in wild type and various mutant infections, and the requirement for homologous DNA segments will be further explored. Direct studies of T4 recombination will also utilize site-specific double-strand breaks in the phage genome. A single-strand annealing model for genetic recombination has long been postulated for phage T4 and has recently emerged for recombination between repeated eukaryotic genes. This model will be tested by analyzing the recombination of DNA molecules that could provide complementary single-stranded regions adjacent to double-strand breaks. The mechanism of recombination at native T4 recombination hotspots will also be examined, particularly because these hotspots are triggered by the above-mentioned replication origins of the virus.