Staphylococcal self-loading helicases couple the staircase mechanism with inter domain high flexibility

Abstract

Replication is a crucial cellular process. Replicative helicases unwind DNA providing the template strand to the polymerase and promoting replication fork progression. Helicases are multi-domain proteins which use an ATPase domain to couple ATP hydrolysis with translocation, however the role that the other domains might have during translocation remains elusive. Here, we studied the unexplored self-loading helicases called Reps, present in Staphylococcus aureus pathogenicity islands (SaPIs). Our cryoEM structures of the PriRep5 from SaPI5 (3.3 Å), the Rep1 from SaPI1 (3.9 Å) and Rep1–DNA complex (3.1Å) showed that in both Reps, the C-terminal domain (CTD) undergoes two distinct movements respect the ATPase domain. We experimentally demonstrate both in vitro and in vivo that SaPI-encoded Reps need key amino acids involved in the staircase mechanism of translocation. Additionally, we demonstrate that the CTD′s presence is necessary for the maintenance of full ATPase and helicase activities. We speculate that this high interdomain flexibility couples Rep′s activities as initiators and as helicases.

Structural and functional insights into the helicase protein E5 of Mpox virus

“…Like MPXV E5, the C-Tail domain is also present in many other SF3 family members, such as SaPI5 PriRep1 36 , Cyanophage S-2L PrimPol, Sulfolobus islandicus pRN1, ASFV C962R, and NrS-1 PrimPol. As demonstrated by mutagenesis and/or structural studies, the C-Tail is important for domain arrangement and DNA unwinding by ASFV C962R 33 and NrS-1 PrimPol 35 .…”

SV40 T-antigen uses a DNA shearing mechanism to initiate origin unwinding

“…The key elements of the T-Ag origin recognition and unwinding system are conserved in a group of mobile DNA elements first identified in Staphylococcus aureus known as SaPI’s. The structure of the SaPI origins mirrors that of SV40, with repeat sequences arranged in opposite orientations that recruit and direct the self-loading of two head-to-head homo-hexameric replicative helicases ( 72 – 74 ). Hence, besides the use of double hexamers to initiate dsDNA unwinding in eukaryotes and archaea, it may also occur in some bacterial settings.…”

Mobile genetic element-encoded putative DNA primases composed of A-family polymerase—SSB pairs

“…The ORF between the putative Pol and helicase ORFs was assigned as a putative SSB (MP homolog) if it was predicted to have an OB fold—this was true for all cases in Table 1 except Geobacillus vulcani , in which this ORF was absent. Most of the helicases associated with these PolA-SSB primases were annotated as containing COG3378 and/or the D5_N and Primase C_term conserved domains, as are the helicases associated with CcPol-MP pairs in many SCC elements and the “Rep” helicase from SaPI5 ( Qiao et al, 2022 ). Despite frequent mis-annotation as “primase” it is important to note that these helicases do not contain primase domains—they are termed “primase C_term” because DnaG- and AEP-family primases are often found upstream of or N-terminally fused to helicases of this family.…”

“…Immediately preceding the recombinase operon is one that, while variable, always encodes a helicase ( Mir-Sanchis et al, 2016 ). Related helicases (generally called “rep”) are found in the better-understood staphylococcal pathogenicity islands (SaPI), which are known to replicate as part of their life cycle ( Fillol-Salom et al, 2018 ; Qiao et al, 2022 ). In the SaPIs, the helicase is always preceded by, and sometimes fused to, a primase.…”