Initiation of DNA Replication

Abstract

In recent years it has become clear that complex regulatory circuits control the initiation step of DNA replication by directing the assembly of a multicomponent molecular machine (the orisome) that separates DNA strands and loads replicative helicase at oriC , the unique chromosomal origin of replication. This chapter discusses recent efforts to understand the regulated protein-DNA interactions that are responsible for properly timed initiation of chromosome replication. It reviews information about newly identified nucleotide sequence features within Escherichia coli oriC and the new structural and biochemical attributes of the bacterial initiator protein DnaA. It also discusses the coordinated mechanisms that prevent improperly timed DNA replication. Identification of the genes that encoded the initiators came from studies on temperature-sensitive, conditional-lethal mutants of E. coli , in which two DNA replication-defective phenotypes, "immediate stop" mutants and "delayed stop" mutants, were identified. The kinetics of the delayed stop mutants suggested that the defective gene products were required specifically for the initiation step of DNA synthesis, and subsequently, two genes, dnaA and dnaC , were identified. The DnaA protein is the bacterial initiator, and in E. coli , the DnaC protein is required to load replicative helicase. Regulation of DnaA accessibility to oriC , the ordered assembly and disassembly of a multi-DnaA complex at oriC , and the means by which DnaA unwinds oriC remain important questions to be answered and the chapter discusses the current state of knowledge on these topics.

Chromosome replication, cell growth, division and shape: a personal perspective

“…Combining the noted constancy of C and D values ( Helmstetter et al, 1968 ) with the way mean cell mass change with τ ( Schaechter et al, 1958 ) resulted in an important insight: cell mass M i at the time of replication-initiation is roughly constant per replication origin oriC ( Donachie, 1968 ; Pritchard, 1968 ; Pritchard et al, 1969 ). The molecular mechanism regulating initiation of replication, occurring synchronously from all existing oriC copies and once per cell cycle, is under investigation (e.g., Leonard and Grimwade, 2010 ), but the apparent constancy of the Mi / oriC ratio is very useful, conferring a quantitative description of the bacterial cell. The cycle ends C + D min after initiation, when cell mass reaches M i × 2 ( C + D )/ τ .…”

Random versus Cell Cycle-Regulated Replication Initiation in Bacteria: Insights from Studying Vibrio cholerae Chromosome 2

“…The dnaA autorepression circuit is more complex than that found in plasmids, as there are two promoters and a multitude of operators to which DnaA binds (33)(34)(35). Additionally, the promoter region is under methylation control, as discussed below for sequestration (36).…”

DNA gyrase activity regulates DnaA‐dependent replication initiation in Bacillus subtilis

“…One critical factor in replication initiation and progression is DNA topology (Smelkova and Marians, ; Leonard and Grimwade, ; Magnan and Bates, ). Much of what is known regarding the role of supercoiling in bacterial replication comes from in vitro studies that use plasmid‐based systems and proteins purified from E. coli (Kaguni et al ., ; Funnell et al ., ; Kornberg, ; Hiasa and Marians, ; Smelkova and Marians, ).…”

“…Oligomerization and cooperative binding of DnaA leads to the melting of the origin at the DNA unwinding element (DUE), and subsequent replisome assembly. Regulation of replication is important for proper cell proliferation and generally occurs at the initiation step through modulation of DnaA binding and activity (Boye et al, 2000;Katayama et al, 2010;Leonard and Grimwade, 2010).…”