Initiation of DNA replication

长期以来，T4噬菌体一直是研究DNA复制、转录和重组的模型，其相对较小的尺寸和定义良好的生物化学使其成为将体外生物化学整合到染色体环境中的优秀模式生物。作为描述T4感染期间染色体交易动态的第一步，我们采用了一套独特的宏观阵列策略来识别病毒DNA合成的来源，并实时监控新生DNA在整个基因组中的实际积累。我们发现，T4DNA合成来自单个感染细胞群体中至少5个离散的基因座，分别位于Oria、Oric、Orie、Orif和orig附近。这是T4感染细胞的单个群体中存在多个活跃来源的第一个直接证据，这意味着T4将为其他多来源系统提供洞察，如人类。起源基因产生的新生DNA在空间和时间上受到调控，导致在起源附近积累多个短DNA，这些DNA后来延伸到全长基因组。这种复制模式似乎对染色体功能至关重要，改变复制动态的突变也会改变整个基因组的转录模式，这表明这两个过程是相互依赖的。事实上，当转录在感染后被阻止时，用于启动复制的起始点就会改变。据推测，复制和转录之间的这种相互作用已经塑造了T4基因组，我们的工作为未来描述有助于T4基因组功能的分子动力学奠定了基础，并为多起源染色体的进化提供了见解。

The bacteriophage T4 has long served as a model for the study of DNA replication, transcription, and recombination, and its relatively small size and well defined biochemistry make it an excellent model organism in which to integrate in vitro biochemistry into a chromosomal context. As a first step in characterizing the dynamics of chromosomal transactions during T4 infection, we employed a unique set of macro array strategies to identify the origins of viral DNA synthesis and monitor the actual accumulation of nascent DNA across the genome in real time. We have found that T4 DNA synthesis originates from at least five discrete loci within a single population of infected cells, near oriA, oriC, oriE, oriF, and oriG. This is the first direct evidence of multiple active origins within a single population of T4-infected cells, and it implies that T4 will provide insights into other multi-origin systems, like humans. The nascent DNAs produced from origin loci are regulated spatially and temporally, leading to the accumulation of multiple, short DNAs near the origins, which are later extended to full length genomes. This pattern of replication appears pivotal to the chromosome function, and mutations that alter replication dynamics also alter transcription patterns across the genome, suggesting that the two processes are dependent on one another. Indeed, when transcription is prevented at later times during infection, the origins used to initiate replication change. Presumably this interplay between replication and transcription has shaped the T4 genome, and our work provides the foundation for the future characterization of the molecular dynamics that contribute to T4 genome function and provide insights into the evolution of multi origin chromosomes.