Properties of the PriA Helicase Domain and Its Role in Binding PriA to Specific DNA Structures

Hua-Wei Chen,Stella H North,Hiroshi Nakai

doi:10.1074/jbc.m404769200

Hua-Wei Chen, Stella H North + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m404769200

Copy DOI

Abstract

Primosome assembly protein PriA functions in the assembly of the replisome at forked DNA structures. Whereas its N-terminal DNA binding domain (DBD) binds independently to DNA, the affinity of DBD protein for forked structures is relatively weak. Although the PriA helicase domain (HD) is required for high affinity fork binding, HD protein had very low affinity for DNA. It had only low levels of ATPase activity, and it hydrolyzed ATP when DNA was absent whereas PriA did not. HD catalyzed unwinding of a minimal substrate composed of a duplex with a 3' single-stranded tail. Single-strand binding protein (SSB) bound to the tail of this substrate inhibited this reaction by full-length PriA but enhanced the reaction by HD. SSB stabilized binding of PriA but not of DBD or HD to duplexes with a 5' or 3' single-stranded tail. On forked substrates SSB enhanced helicase action on the lagging-strand arm by PriA but not by HD. The results indicate that synergy of the DBD and HD allows stable binding at the interface between duplex and single-stranded DNA bound by SSB. This mode of binding may be analogous to fork binding, which orients the helicase to act on the lagging-strand side of the fork.

Highlights

Escherichia coli PriA protein is one of seven proteins that make up the restart primosome, an apparatus that promotes assembly of replisomes at recombination intermediates and stalled replication forks
The results indicate that synergy of the DNA binding domain (DBD) and helicase domain (HD) allows stable binding at the interface between duplex and single-stranded DNA bound by Singlestrand binding protein (SSB)
Purification of DBD and HD of PriA—The hypersensitive trypsin cleavage site at Arg-198 divides PriA protein into two domains (Fig. 1A), the DBD and HD. To purify these parts of the PriA protein, DBD protein was expressed with an intein tag attached at the C terminus, and HD was expressed with the intein tag at the N terminus

Summary

Function of the PriA Helicase Domain

Not inhibit helicase action on the lagging-strand arm even when it is directly bound to this arm (22). These results have suggested that fork binding orients the HD to act on the lagging-strand arm, SSB bound to the fork enhancing this mode of PriA binding. We report that the HD has ATPase activity and can carry out the basal mode of helicase action. It has very low DNA binding affinity and, has very low activity. In full-length PriA, the HD plays an important role together with the DBD in recognizing the transition from duplex to single-stranded DNA, most likely the bend between the parental duplex and the leading- and lagging-strand arms of the fork

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION