A phage‐encoded inhibitor of Escherichia coli DNA replication targets the DNA polymerase clamp loader

Abstract

SummaryColiphage N4 infection leads to shut-off of host DNA replication without inhibition of host transcription or translation. We report the identification and characterization of gp8, the N4 gene product responsible for this phenotype. N4 gp8 is an Escherichia coli bacteriostatic inhibitor that colocalizes with the E. coli replisome in a replication-dependent manner. Gp8 was purified and observed to cross-link to complexes containing the replicative DNA polymerase, DNAP III, in vivo. Purified gp8 inhibits DNA polymerization by DNA polymerase III holoenzyme in vitro by interfering with polymerase processivity. Gp8 specifically inhibits the clamp-loading activity of DNAP III by targeting the delta subunit of the DNAP III clamp loader; E. coli mutations conferring gp8 resistance were identified in the holA gene, encoding delta. Delta and gp8 interact in vitro; no interaction was detected between gp8 inactive mutants and wild-type delta or between delta gp8-resistant mutants and wild-type gp8. Therefore, this work identifies the DNAP III clamp loader as a new target for inhibition of bacterial growth. Finally, we show that gp8 is not essential in N4 development under laboratory conditions, but its activity contributes to phage yield.

Promoter-Specific Transcription Inhibition in Staphylococcus aureus by a Phage Protein

“…DNA replication is often a target of early phage proteins (Datta et al, 2005; Yano and Rothman-Denes, 2011). However, expression of genes involved in replication (Xu et al, 2010) was not significantly altered in the presence of gp67 in vivo (Figure 3B).…”

Bacterial Virus Ontology; Coordinating across Databases

“…Other mechanisms redirect bacterial metabolic pathways to the bacterial virus reproduction cycle. Through “Inhibition of host DNA replication”, viruses prevent host replication and division, thereby improving available dNTPs and metabolic activity for their own replication [50,51]. …”

Influenza virus matrix protein M1 interacts with SLD5 to block host cell cycle

“…We found that M1 blocks host cell DNA replication likely by interacting with SLD5 to disrupt the normal function of the GINS complex. Some bacterial virus can shut off host DNA replication to arrest host growth (Belley et al, 2006;Seco et al, 2013;Yano & Rothman-Denes, 2011). First, knockdown or mutation of SLD5 (△7aa) in cells resulted in delayed cell cycle progress.…”

“…Several pieces of evidence support this founding. Bacteriophage N4, for example, is known to shut off host DNA replication, whereas the host genome remains structurally intact (Yano & Rothman-Denes, 2011). It has been reported that SLD5 interacts with Psf1, Psf2, and Psf3 (Choi et al, 2007;Gouge & Christensen, 2010;Joshi et al, 2016;Takayama et al, 2003).…”

“…The SLD5△7aa mutant failed to bind Psf2, which might Inhibiting host replication provides an advantage during virus life cycle by reducing or eliminating competing host macromolecular synthesis (Chen & Makino, 2004;Flemington, 2001). Some bacterial virus can shut off host DNA replication to arrest host growth (Belley et al, 2006;Seco et al, 2013;Yano & Rothman-Denes, 2011). Bacteriophage N4, for example, is known to shut off host DNA replication, whereas the host genome remains structurally intact (Yano & Rothman-Denes, 2011).…”