Mensa-Wilmot K, Carroll K, McMacken R
Department of Biochemistry, Johns Hopkins University, School of Hygiene and Public Health, Baltimore, MD 21205.
EMBO J. 1989 Aug;8(8):2393-402. doi: 10.1002/j.1460-2075.1989.tb08369.x.
Initiation of bacteriophage lambda DNA replication in vivo and in crude in vitro systems is strongly dependent on transcription at or near the lambda replication origin (ori lambda). Through its capacity to prevent RNA polymerase-mediated 'transcriptional activation' of lambda DNA replication, the lambda cI repressor is capable of negatively regulating initiation of lambda DNA replication, even when all required replication proteins are present. Surprisingly, the strict requirement for transcriptional activation of lambda DNA replication was lost when lambda replication was initiated in an in vitro system composed of nine purified replication proteins [Mensa-Wilmot et al. (1989) J. Biol. Chem., 264, 2853-2861]. We have found that crude extracts of Escherichia coli contain proteins that are capable of restoring the physiological linkage between transcription and ori lambda-dependent replication when they are added to the nine-protein replication system. The protein primarily responsible for this effect has been purified and identified as protein HU, a histone-like protein that is a major constituent of the bacterial nucleoid. HU, when present at a 1:1 weight ratio with supercoiled ori lambda plasmid, is a potent inhibitor of lambda DNA replication in the nine-protein replication system. However, when the ori lambda template is transcribed by E. coli RNA polymerase, the HU-mediated inhibition of lambda DNA replication is abolished. HU does not inhibit propagation of lambda replication forks. Instead, HU apparently interferes with the assembly or function of nucleoprotein structures containing the E. coli DnaB helicase that are formed at ori lambda prior to priming and DNA synthesis. We suggest that the chromatin structure of the template DNA in the region surrounding ori lambda plays a central role in the negative regulation of the initiation of lambda DNA replication in vivo.
噬菌体λ DNA 在体内和粗制体外系统中的复制起始强烈依赖于λ复制起点（ori λ）处或其附近的转录。λ cI 阻遏物能够阻止 RNA 聚合酶介导的λ DNA 复制的“转录激活”，即使所有必需的复制蛋白都存在，它也能够负向调节λ DNA 复制的起始。令人惊讶的是，当在由九种纯化的复制蛋白组成的体外系统中起始λ复制时，对λ DNA 复制转录激活的严格要求丧失了[门萨 - 威尔莫特等人（1989 年）《生物化学杂志》，264，2853 - 2861]。我们发现大肠杆菌的粗提物中含有一些蛋白质，当将它们添加到九蛋白复制系统中时，能够恢复转录与 ori λ 依赖性复制之间的生理联系。对这种效应起主要作用的蛋白质已被纯化并鉴定为 HU 蛋白，它是一种类组蛋白，是细菌类核的主要成分。当 HU 与超螺旋 ori λ 质粒以 1:1 的重量比存在时，它是九蛋白复制系统中λ DNA 复制的有效抑制剂。然而，当 ori λ 模板由大肠杆菌 RNA 聚合酶转录时，HU 介导的λ DNA 复制抑制作用就会被消除。HU 并不抑制λ复制叉的延伸。相反，HU 显然干扰了在引发和 DNA 合成之前在 ori λ 处形成的含有大肠杆菌 DnaB 解旋酶的核蛋白结构的组装或功能。我们认为，ori λ 周围区域模板 DNA 的染色质结构在体内λ DNA 复制起始的负调控中起核心作用。
