Primase is required for helicase activity and helicase alters the specificity of primase in the enteropathogen Clostridium difficile

Abstract

DNA replication is an essential and conserved process in all domains of life and may serve as a target for the development of new antimicrobials. However, such developments are hindered by subtle mechanistic differences and limited understanding of DNA replication in pathogenic microorganisms. Clostridium difficile is the main cause of healthcare-associated diarrhoea and its DNA replication machinery is virtually uncharacterized. We identify and characterize the mechanistic details of the putative replicative helicase (CD3657), helicase-loader ATPase (CD3654) and primase (CD1454) of C. difficile, and reconstitute helicase and primase activities in vitro. We demonstrate a direct and ATP-dependent interaction between the helicase loader and the helicase. Furthermore, we find that helicase activity is dependent on the presence of primase in vitro. The inherent trinucleotide specificity of primase is determined by a single lysine residue and is similar to the primase of the extreme thermophile Aquifex aeolicus. However, the presence of helicase allows more efficient de novo synthesis of RNA primers from non-preferred trinucleotides. Thus, loader–helicase–primase interactions, which crucially mediate helicase loading and activation during DNA replication in all organisms, differ critically in C. difficile from that of the well-studied Gram-positive Bacillus subtilis model.

Genome Location Dictates the Transcriptional Response to PolC Inhibition in Clostridium difficile

“…Such a plot places the terC region around 2.2 Mb from CD0001, near the CD1931 (CD630DERM_RS10465) open reading frame ( Fig. 4A) (34). We noted that the differential expression appeared to correlate with genome location (Tables 2, 3, and S2), as many of the upregulated genes have either low or high gene identifiers (CD numbers) indicative of an origin proximal location; conversely, many of the downregulated genes appear to be located away from oriC.…”

Identification of the Unwinding Region in the Clostridioides difficile Chromosomal Origin of Replication

“…B. subtilis DnaB protein also affects the DNA topology and has been shown to be important for recruiting oriC to the membrane ( Rokop et al, 2004 ; Zhang et al, 2005 ). C. difficile lacks a homolog for the DnaB protein, although the closest homolog of the DnaD protein (CD3653) ( van Eijk et al, 2017 ) may perform similar functions in the origin remodeling ( van Eijk et al, 2016 ). Direct interaction of DnaA-DnaD through the DnaA Domain I was structurally determined and the residues present at the interface were solved ( Martin et al, 2018 ).…”

“…In C. difficile , knowledge on DNA replication is limited. Though many proteins appear to be conserved between well-characterized species and C. difficile , only certain replication proteins have been experimentally characterized for C. difficile ( Torti et al, 2011 ; Briggs et al, 2012 ; van Eijk et al, 2016 ). DNA polymerase C (PolC, CD1305) of C. difficile has been studied in the context of drug-discovery and appears to have a conserved primary structure similar to other low-[G + C] gram-positive organisms ( Torti et al, 2011 ).…”

“…It is inhibited in vitro and in vivo by compounds that compete for binding with dGTP ( van Eijk et al, 2019 ; Xu et al, 2019 ). Helicase (CD3657), essential for DNA duplex unwinding, was found to interact in an ATP-dependent manner with a helicase loader (CD3654) and loading was proposed to occur through a ring-maker mechanism ( Davey and O’Donnell, 2003 ; van Eijk et al, 2016 ). However, in contrast to helicase of the Firmicute Bacillus subtilis , C. difficile helicase activity is dependent on activation by the primase protein (CD1454), as has also been described for Helicobacter pylori ( Bazin et al, 2015 ; van Eijk et al, 2016 ).…”

“…Helicase (CD3657), essential for DNA duplex unwinding, was found to interact in an ATP-dependent manner with a helicase loader (CD3654) and loading was proposed to occur through a ring-maker mechanism ( Davey and O’Donnell, 2003 ; van Eijk et al, 2016 ). However, in contrast to helicase of the Firmicute Bacillus subtilis , C. difficile helicase activity is dependent on activation by the primase protein (CD1454), as has also been described for Helicobacter pylori ( Bazin et al, 2015 ; van Eijk et al, 2016 ). C. difficile helicase stimulates primase activity at the trinucleotide 5′d(CTA), but not at the preferred trinucleotide 5′-d(CCC) ( van Eijk et al, 2016 ).…”