Kinetics of DNA Unwinding by the RecD2 Helicase from Deinococcus radiodurans

Abstract

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)....

Oxidative Stress Resistance inDeinococcus radiodurans

“…RecD2 (RecD present in genomes lacking RecBC) differs from RecD1 by an N-terminal extension (521) and represents an ancestral form of RecD (429). Deinococcal RecD has a helicase activity with 5Ј-to-3Ј polarity and low processivity (556,644). Its deletion enhances the efficiency of transformation by exogenous homologous DNA, which suggests that RecD has antirecombinogenic properties in D. radiodurans (551).…”

RecD2 Helicase Limits Replication Fork Stress in Bacillus subtilis

“…1). The RecD2 protein from Deinococcus radiodurans has been shown to be important for resistance to gamma irradiation, hydrogen peroxide, and UV irradiation in vivo (21), and it is able to unwind short hairpin and forked DNA structures in a 5=-to-3= direction (22)(23)(24). A recent genome-wide screen for genes associated with a colony papillation phenotype in Bacillus anthracis identified the gene BAS4289 encoding a RecD2 homolog (20,25).…”

Molecular and Functional Characterization of RecD, a Novel Member of the SF1 Family of Helicases, from Mycobacterium tuberculosis

“…E. coli Rep and UvrD function as dimers, whereas Bacillus stearothermophilus PcrA, phage T4 Dda, and TraI helicases are active in the monomeric form (56 -62). E. coli and D. radiodurans RecD exist in solution as monomers (63)(64)(65). Using multiple complementary methods, we show that MtRecD exists in solution as a homodimer in the absence of DNA.…”