DNA Unwinding Step-size of E.coli RecBCD Helicase Determined from Single Turnover Chemical Quenched-flow Kinetic Studies

115

135

Structural studies of the Bacillus stearothermophilus PcrA protein along with biochemical studies of the single-stranded (ss) DNA translocation activity of PcrA monomers have led to the suggestion that a PcrA monomer possesses processive helicase activity in vitro. Yet definitive studies testing whether the PcrA monomer possesses processive helicase activity have not been performed. Here we show, using single turnover kinetic methods, that monomers of PcrA are able to translocate along ssDNA, in the 3 to 5 direction, rapidly and processively, whereas these same monomers display no detectable helicase activity under the same solution conditions in vitro. The PcrA monomer ssDNA translocation activity, although necessary, is not sufficient for processive helicase activity, and thus the translocase and helicase activities of PcrA are separable. These results also suggest that the helicase activity of PcrA needs to be activated either by self-assembly or through interactions with accessory proteins. This same behavior is displayed by both the Escherichia coli Rep and UvrD monomers. Hence, all three of these SF1 enzymes are ssDNA translocases as monomers but do not display processive helicase activity in vitro unless activated. The fact that the translocase and helicase activities are separable suggests that each activity may be used for different functions in vivo.DNA helicases are nucleic acid motor proteins that use nucleoside triphosphate binding and hydrolysis to unwind duplex DNA (1-4). These enzymes generally also have the ability to translocate with biased directionality along singlestranded (ss) 3 DNA (5-9). In fact, some of the biological activities of these enzymes, such as displacement of other proteins from ssDNA (10 -16), may be dependent only on the ability of these enzymes to translocate along ssDNA rather than on helicase activity. DNA helicases have been grouped into families and superfamilies defined by conserved amino acid sequence motifs (17). These enzymes can also be divided into two distinct structural classes, with one class functioning as toroidal or ringlike hexamers (4). By far, the largest number of helicases and putative helicases belong to the nonhexameric SF1 or SF2 superfamilies (17).SF1 enzymes have often been considered to be monomeric helicases (18,19). Yet direct experimental tests of whether a monomeric protein has helicase activity have been performed for only a few enzymes. The most direct experimental approach to address the question of what oligomeric form of the enzyme is needed for helicase activity requires examination of the DNA unwinding kinetics under single turnover conditions, such that only a single round of DNA binding is allowed. In such an experiment, the DNA substrate is prebound with enzyme under conditions such that the oligomeric form of the enzyme is known (e.g. a monomer), and the reaction is started by the addition of nucleoside triphosphate (e.g. ATP) along with a "trap" for free enzyme (20,21). Any DNA unwinding that is observed in such a single turnover e...

Section: Resultsmentioning

confidence: 99%

Bacillus stearothermophilus PcrA Monomer Is a Single-stranded DNA Translocase but Not a Processive Helicase in Vitro

Niedziela-Majka

Chesnik

Tomko

et al. 2007

115

135

“…Determination of Kinetic Parameters for Unwinding-Unwinding data for substrates of varying duplex length were fit by nonlinear least squares analysis using Scientist (MicroMath Scientific Software) (27)(28)(29). The kinetic step size (m) is defined as the number of base pairs that are unwound before each ratelimiting slow step and can be calculated from Equation 1, where L T is the total length of the duplex, L 0 is the length of DNA that spontaneously melts, and n is the total number of kinetic steps required to unwind this substrate.…”

Section: Methodsmentioning

confidence: 99%

Yeast Pif1 Helicase Exhibits a One-base-pair Stepping Mechanism for Unwinding Duplex DNA

Ramanagoudr-Bhojappa

Chib

Byrd

et al. 2013

“…RecD2 DNA Unwinding Mechanism-A number of previous studies show that DNA helicases require multiple steps for complete unwinding of short dsDNA substrates (23,27,28). The kinetic step size of a helicase is the number of base pairs unwound between two successive rate-limiting (kinetically significant) steps in the unwinding cycle (29).…”

mentioning

confidence: 99%

“…TraI, which at 192 kDa is much larger than either RecD2 or Dda, unwinds DNA very rapidly (1100 bp/sec) and with high processivity, and has a 6 -8 bp step size (40). Unwinding rates for other SF1 helicases that are more distantly related to RecD2 vary from 30 bp/s for PcrA (32)) and 68 bp/s for UvrD (41)), to 790 bp/s for RecBCD (28). However, the kinetic step sizes of these enzymes are similar to that of RecD2: UvrD (4 -5 bp/step (27)), PcrA (4 bp/step (35)), and RecBC and RecBCD (ϳ3-4 bp/step (28,37)).…”

mentioning

confidence: 99%

“…Unwinding rates for other SF1 helicases that are more distantly related to RecD2 vary from 30 bp/s for PcrA (32)) and 68 bp/s for UvrD (41)), to 790 bp/s for RecBCD (28). However, the kinetic step sizes of these enzymes are similar to that of RecD2: UvrD (4 -5 bp/step (27)), PcrA (4 bp/step (35)), and RecBC and RecBCD (ϳ3-4 bp/step (28,37)). The similarity in kinetic step size suggests an underlying mechanistic similarity in DNA unwinding by these helicases, despite the differences in their unwinding rates.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Kinetics of DNA Unwinding by the RecD2 Helicase from Deinococcus radiodurans

Shadrick

Julin

2010

RecD2 from Deinococcus radiodurans is a superfamily 1 DNA helicase that is homologous to the Escherichia coli RecD protein but functions outside the context of RecBCD enzyme. We report here on the kinetics of DNA unwinding by RecD2 under single and multiple turnover conditions. There is little unwinding of 20-bp substrates by preformed RecD2-dsDNA complexes when excess ssDNA is present to trap enzyme molecules not bound to the substrate. A shorter 12-bp substrate is unwound rapidly under single turnover conditions. The 12-bp unwinding reaction could be simulated with a mechanism in which the DNA is unwound in two kinetic steps with rate constant of k unw ‫؍‬ 5.5 s ؊1 and a dissociation step from partially unwound DNA of Deinococcus radiodurans is a Gram-positive bacterium that is extremely resistant to the lethal effects of ionizing radiation, ultraviolet light, oxidants, and dessication. It is widely studied as a model for organisms that are highly resistant to these agents, and for general understanding of how these agents affect living organisms. There has been much recent work on the physiological attributes, enzymes, and enzymatic pathways that contribute to its extraordinary ability to survive under extreme conditions (reviewed in Refs. 1-3). Double-strand DNA breaks (DSBs) 2 can arise in cells after treatment with ionizing radiation, oxidizing agents, and during desiccation (4), and they are lethal if unrepaired. D. radiodurans has the capacity to recover from hundreds of radiationinduced DSBs (1, 5). The enzymatic machinery that it uses to repair DSBs is not fully understood. DSB repair in E. coli and other bacteria requires the RecBCD helicase/nuclease to initiate the repair by processing broken DNA ends and loading the RecA recombinase (6, 7). DSB repair in D. radiodurans is thought to involve similar processing of the DNA ends (5, 8), and the repair is dependent on RecA (5, 9). However, D. radiodurans lacks homologues of RecB and RecC (10), and so the identity of the helicase(s) and nuclease(s) that process broken DNA ends are not clear. Recent work has indicated that the RecJ exonuclease and other enzymes of the RecF pathway may be important for DSB repair in D. radiodurans (11,12).Interestingly, D. radiodurans does have a homologue of the RecD subunit of the RecBCD enzyme found in Escherichia coli and other bacteria (10). Sequence analysis shows that the D. radiodurans RecD shares with the RecD subunits seven short amino acid motifs that are conserved in Superfamily 1 (SF1) DNA and RNA helicases (13). However, the D. radiodurans protein is larger than that from E. coli, with an N-terminal extension whose amino acid sequence is poorly conserved. For this reason, the D. radiodurans RecD protein, and close protein relatives found in a number of other bacteria, have been named RecD2 (14). The in vivo function of D. radiodurans RecD2 is not clear, but recD2 mutant cells are 100 -1000 times more sensitive than wild type to the cytotoxic effect of UV light, gamma irradiation, and hydrogen peroxide (15-17)....