Crystal Structures of Complexes of PcrA DNA Helicase with a DNA Substrate Indicate an Inchworm Mechanism

Abstract

We have determined two different structures of PcrA DNA helicase complexed with the same single strand tailed DNA duplex, providing snapshots of different steps on the catalytic pathway. One of the structures is of a complex with a nonhydrolyzable analog of ATP and is thus a "substrate" complex. The other structure contains a bound sulphate ion that sits in a position equivalent to that occupied by the phosphate ion produced after ATP hydrolysis, thereby mimicking a "product" complex. In both complexes, the protein is monomeric. Large and distinct conformational changes occur on binding DNA and the nucleotide cofactor. Taken together, these structures provide evidence against an "active rolling" model for helicase action but are instead consistent with an "inchworm" mechanism.

Chromatin remodeling: insights and intrigue from single-molecule studies

“…Crystal structures, biochemical studies and single-molecule analyses of both SF1-and SF2-family translocases (PcrA, RecG, UvrD, NS3 and others) have provided many important insights into these issues 39,40,[57][58][59] . Currently, there is no crystal structure of the ATPase domain of a nucleosome-binding remodeler (SWI/SNF or ISWI), but recent structures of the SWI/SNF-family member Rad54 (with DNA) show remarkable structural homology to the SF2 family of RNA and DNA translocases (see Fig.…”

“…The DNAtracking domain has two RecA-like motifs, and between them is a pocket for nucleotide binding as well as a platform for DNA interaction. Nucleotide binding and hydrolysis induce conformational changes between the two RecA-like motifs that result in the net movement of the tracking domain 1 bp in the 3′-to-5′ direction along one of the DNA strands, termed the tracking strand 58,61 . The DBD binds nucleic acid in front of the DNA-tracking domain and undergoes a conformational change that is coordinated with those of the tracking domain.…”

“…An alternative (but similar) mechanism starts with the DBD already bound at an advanced position, from which it then actively pulls DNA toward the tracking domain, essentially feeding nucleic acid substrate into the tracking domain for subsequent tracking. Once the tracking domain tracks through this DNA, the DBD then steps forward, completing the inchworm cycle 58 . The key difference between these two similar models is whether the DBD in front of the tracking is a passive DNA anchor or whether it instead provides an active DNA-pulling force that feeds DNA into the tracking domain.…”

Erratum to: Structure and Mechanisms of SF1 DNA Helicases

“…SF1 includes three families (Rep/UrvD, Pif1/RecD, and Upf1 like) [86] and can be divided into groups based on the direction of translocation on ssDNA: 3 -5 for SF1A helicases and 5 -3 for SF1B helicases [43]. Some of the well-characterized SF1A helicases are PcrA, Rep, and UvrD T [87][88][89][90][91][92]. Well-characterized members of SF1B include RecD and Dda [93,94] (He et al, in press).…”

Flaviviral helicase: Insights into the mechanism of action of a motor protein

“…F-Hel is composed of tree domains (I-III), each spanning about 140-160 residues. The first two domains display a characteristic 'RecA-like' fold [2], composed of a b-sheet hosting six parallel b-strands (topology 1-6-5-2-4-3) surrounded by five a-helices; moreover, domain II contains a b-hairpin protruding toward domain III (located between b-strands 5 and 6). The third domain is mainly built by six antiparallel a-helices.…”