Demonstration of Unidirectional Single-Stranded DNA Translocation by PcrA Helicase:  Measurement of Step Size and Translocation Speed

Abstract

Using a fluorescent sensor for inorganic phosphate, the kinetics of ATP hydrolysis by PcrA helicase were measured in the presence of saturating concentrations of oligonucleotides of various lengths. There is a rapid phase of inorganic phosphate release that is equivalent to several turnovers of the ATPase, followed by slower steady-state ATP hydrolysis. The magnitude of the rapid phase is governed by the length of single-stranded DNA, while the slow phase is independent of its length. A kinetic model is presented in which the rapid phase is associated with translocation along single-stranded DNA, after the PcrA binds randomly along the DNA. There is a linear relationship between the length of single-stranded DNA and both the duration and amplitude of the rapid phase. These data suggest that the translocation activity occurs at 50 bases/s in unidirectional single-base steps, each requiring the hydrolysis of 1 ATP molecule.

A Motor that Makes Its Own Track: Helicase Unwinding of DNA

“…Experimentally measured values are typically smaller, usually 0.33-1 bp unwound/ATP hydrolyzed [2]. For PcrA, the measured efficiency of unwinding is approximately 1 bp unwound/ATP [11].…”

“…Helicases offer a particularly appealing choice for a simplified physical description because they are an important class of enzyme which interacts with NA, yet they have many relatively simple features. For example, many helicases have unwinding rates which are independent of the NA sequence unwound [2,11]. Thus, the information content of the NA does not play an essential role in helicase operation-a large simplification compared to RNA polymerases, for example [12,13].…”

Chromatin remodeling: insights and intrigue from single-molecule studies

“…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.…”

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).…”

“…Oligomerization can affect their NTPase, DNA-binding and -unwinding activities [45]. The monomeric forms of some SF1 enzymes, Rep [119,120,147,154,155], UvrD [121,134,156], and PcrA [89,91,127,145], are processive translocases [91,92,156,157], but do not display DNA unwinding activity in vitro [147]. On the other hand, the SF1 helicases Dda [158] and TraI [159] are able to function as monomeric helicases in vitro, b.…”