Duplex opening by primosome protein PriA for replisome assembly on a recombination intermediate.
PriA and other primosome assembly proteins of Escherichia coli recruit the major replicative helicase DnaB for replisome assembly during bacteriophage Mu transposition and replication. MuA transposase catalyzes the transfer of Mu ends to target DNA, forming a potential replication fork that provides the assembly site for the replisome. However, this fork lacks the single-stranded DNA needed to load DnaB. Although no pre-existing primosome assembly sites that bind PriA were found within the Mu end sequences, PriA was able to bind to the forked DNA structure created by MuA. The helicase activity of PriA could then open the duplex to create the DnaB binding site. In a tightly coupled reaction on synthetic forked substrates, PriA promoted both the unwinding of the lagging strand arm and preprimosome assembly to load DnaB onto the lagging strand template. PriA apparently translocated 3' to 5' along the lagging strand template until sufficient single-stranded DNA was exposed for binding of DnaB, which then translocated 5' to 3' in the opposite direction. Mutant PriA lacking helicase activity was unable to promote this process, and loss of PriA helicase impaired Mu DNA replication in vivo and in vitro. This suggests that the opening of the duplex by PriA helicase is a critical step in the initiation of Mu DNA replication. Concerted helicase and primosome assembly functions would allow PriA to act as initiator on recombination intermediates and stalled replication forks. As part of the replisome, PriA may act as a mobile initiator that minimizes interruptions in chromosomal replication.
Jones
J M
JM
Department of Biochemistry and Molecular Biology, Georgetown University Medical Center, 3900 Reservoir Rd NW, Washington, DC, 20007, USA.
Nakai
H
H
D001426:Bacterial Proteins; D010583:Bacteriophage mu / Q000737:chemistry / Q000235:genetics; D001483:Base Sequence; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism*; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D053488:DnaB Helicases; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009692:Nucleic Acid Heteroduplexes / Q000737:chemistry / Q000378:metabolism*; D011995:Recombination, Genetic; D051716:Replication Protein A
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D009692:Nucleic Acid Heteroduplexes; D051716:Replication Protein A; D004265:DNA Helicases; D053488:DnaB Helicases