Escherichia coli PriA helicase: Fork binding orients the helicase to unwind the lagging strand side of arrested replication forks

Escherichia coli PriA is a primosome assembly protein with 3' to 5' helicas e activity whose apparent function is to promote resumption of DNA synthesi s following replication-fork arrest. Here, we describe how initiation of he licase activity on DNA forks is influenced by both fork structure and by si ngle-strand DNA-binding protein. PriA could recognize and unwind forked sub strates where one or both arms were primarily duplex, and PriA required a s mall (two bases or larger) single-stranded gap at the fork in order to init iate unwinding. The helicase was most active on substrates with a duplex la gging-strand arm and a single-stranded leading-strand arm. On this substrat e, PriA was capable of translocating on either the leading or lagging stran ds to unwind the duplex ahead of the fork or the lagging-strand duplex, res pectively. Fork-specific binding apparently orients the helicase domain to unwind the lagging-strand duplex. Binding of single-strand-binding protein to forked templates could inhibit unwinding of the duplex ahead of the fork but not unwinding of the lagging-strand duplex or translocation on the lag ging-strand template. While single-strand-binding protein could inhibit bin ding of PriA to the minimal, unforked DNA substrates, it could not inhibit PriA binding to forked substrates. In the cell, single-strand-binding prote in and fork structure may direct PriA helicase to translocate along the lag ging-strand template of forked structures such that the primosome is specif ically assembled on that DNA strand. (C) 2001 Academic Press.