RepD-mediated recruitment of PcrA helicase at the Staphylococcus aureus pC221 plasmid replication origin, oriD

Abstract

Plasmid encoded replication initiation (Rep) proteins recruit host helicases to plasmid replication origins. Previously, we showed that RepD recruits directionally the PcrA helicase to the pC221 oriD, remains associated with it, and increases its processivity during plasmid unwinding. Here we show that RepD forms a complex extending upstream and downstream of the core oriD. Binding of RepD causes remodelling of a region upstream from the core oriD forming a ‘landing pad’ for the PcrA. PcrA is recruited by this extended RepD–DNA complex via an interaction with RepD at this upstream site. PcrA appears to have weak affinity for this region even in the absence of RepD. Upon binding of ADPNP (non-hydrolysable analogue of ATP), by PcrA, a conformational rearrangement of the RepD–PcrA–ATP initiation complex confines it strictly within the boundaries of the core oriD. We conclude that RepD-mediated recruitment of PcrA at oriD is a three step process. First, an extended RepD–oriD complex includes a region upstream from the core oriD; second, the PcrA is recruited to this upstream region and thirdly upon ATP-binding PcrA relocates within the core oriD.

The structure and function of an RNA polymerase interaction domain in the PcrA/UvrD helicase

“…This multi-functionality is reflected in the ability of UvrD/PcrA to interact physically and functionally with many different partner proteins including UvrB, MutL, MutS, RecA and RepC/D (13–20). We and others have recently shown that PcrA/UvrD also interacts with RNA polymerase (RNAP) (1,14,21), and this interaction could be important for the UvrD-dependent backtracking of stalled RNA polymerase (1).…”

Engineering of a superhelicase through conformational control

“…One such partner of PcrA is RepD, a plasmid replication initiator protein that recognizes and forms a covalent adduct with the oriD sequence of the plasmid, and then recruits PcrA for processive unwinding (24, 25). We prepared an oriD DNA-RepD adduct, and measured the intramolecular conformation of PcrA bound to this adduct (18).…”

Plasmid Rolling-Circle Replication

“…The recruitment of the helicase onto the replication initiation site has also been shown by footprinting experiments using exonuclease (Exo) III (173). RepD binding to oriD creates an area of protection to ExoIII digestion which extends beyond the oriD region: ∼74 to 80 bp upstream of ICR I for the continuous strand and ∼46 to 50 bp downstream of ICR III for the nicked strand (173), although further resistance points can be found within the oriD, after longer digestion times.…”

“…Furthermore, these three stages of protein loading on the DNA have been studied by atomic force microscopy, where RepD appears as a globular particle which bends the DNA fragment around 90°in 39% of the oriD fragments analyzed. However, when both proteins were bound to the DNA, the proportion of bent DNA increased up to 60%, while ADPNP decreased this percentage to 41% (173).…”

“…RepD binding to oriD creates an area of protection to ExoIII digestion which extends beyond the oriD region: ∼74 to 80 bp upstream of ICR I for the continuous strand and ∼46 to 50 bp downstream of ICR III for the nicked strand (173), although further resistance points can be found within the oriD, after longer digestion times. When PcrA was incorporated into the reaction, the helicase was recruited upstream of ICR I, which served to stabilize the complex.…”