DNA Helicase Activity of the RecD Protein from Deinococcus radiodurans

Wang, Jianlei; Julin, Douglas A.

doi:10.1074/jbc.m408645200

Cited by 38 publications

(43 citation statements)

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“…Typical superfamily I helicases, such as UvrD and RecD, can efficiently unwind a duplex DNA with a singlestranded tail of 10 -12 nucleotides (34,35). However, TraI requires at least a 27-nucleotide tail for efficient unwinding and cannot unwind duplex DNA if the tail is shorter than 20 nucleotides (36).…”

Section: Discussionmentioning

confidence: 99%

Functional Characterization of the Multidomain F Plasmid TraI Relaxase-Helicase

Cheng¹,

McNamara²,

Miley³

et al. 2011

Journal of Biological Chemistry

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TraI, a bifunctional enzyme containing relaxase and helicase activities, initiates and drives the conjugative transfer of the Escherichia coli F plasmid. Here, we examined the structure and function of the TraI helicase. We show that TraI binds to singlestranded DNA (ssDNA) with a site size of ϳ25 nucleotides, which is significantly longer than the site size of other known superfamily I helicases. Low cooperativity was observed with the binding of TraI to ssDNA, and a double-stranded DNA-binding site was identified within the N-terminal region of TraI 1-858, outside the core helicase motifs of TraI. We have revealed that the affinity of TraI for DNA is negatively correlated with the ionic strength of the solution. The binding of AMPPNP or ADP results in a 3-fold increase in the affinity of TraI for ssDNA. Moreover, TraI prefers to bind ssDNA oligomers containing a single type of base. Finally, we elucidated the solution structure of TraI using small angle x-ray scattering. TraI exhibits an ellipsoidal shape in solution with four domains aligning along one axis. Taken together, these data result in the assembly of a model for the multidomain helicase activity of TraI.Conjugative plasmid transfer is a central mechanism for the horizontal exchange of genetic material between bacterial cells, as well as for the spread of antibiotic resistance genes and virulence factors (1-3). Conjugative plasmid transfer requires both a DNA relaxase and a DNA helicase. The relaxase initiates DNA transfer by cleaving the transferred strand at a specific site within the oriT, forming a 5Ј-phosphotyrosine intermediate. Following nicking, the helicase uses the energy from ATP hydrolysis to unwind the plasmid and drive the transfer of DNA into the recipient cell. The relaxase completes plasmid transfer by breaking the covalent phosphotyrosine linkage and releasing the transferred DNA for replication in the recipient (2, 4, 5).

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Section: Discussionmentioning

confidence: 99%

Functional Characterization of the Multidomain F Plasmid TraI Relaxase-Helicase

Cheng¹,

McNamara²,

Miley³

et al. 2011

Journal of Biological Chemistry

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“…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.…”

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confidence: 96%

“…The E. coli RecD protein has DNAdependent ATPase-and DNA-unwinding activities (20,21), but it has poor solubility and has been difficult to study in isolation. The D. radiodurans RecD2 is soluble when expressed in E. coli and is relatively easy to purify (13). For this reason, the D. radiodurans RecD2 is a good model for enzymatic study of this novel family of DNA helicases.…”

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confidence: 99%

“…It thus has 5Ј-3Ј polarity and has been placed in the SF1B helicase family (18). The protein appeared to have low processivity, because longer DNAs (52 or 76 bp) were unwound much less efficiently than the 20-bp substrates (13). The three-dimensional structure of RecD2 has been determined, as the free enzyme (18), bound to ssDNA, and bound to ssDNA and an ATP analogue (19).…”

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confidence: 99%

“…The purified D. radiodurans RecD2 protein is a DNA helicase capable of efficient unwinding of 20-bp DNA duplexes with a 5Ј single-stranded extension at least 10 nt in length, or with a forked end (13). It thus has 5Ј-3Ј polarity and has been placed in the SF1B helicase family (18).…”

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confidence: 99%

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Kinetics of DNA Unwinding by the RecD2 Helicase from Deinococcus radiodurans

Shadrick

Julin

2010

Journal of Biological Chemistry

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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)....

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Mechanistic Basis of 5′-3′ Translocation in SF1B Helicases

Saikrishnan

Powell²,

Cook

et al. 2009

Cell

131

210

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Superfamily 1B (SF1B) helicases translocate in a 5'-3' direction and are required for a range of cellular activities across all domains of life. However, structural analyses to date have focused on how SF1A helicases achieve 3'-5' movement along nucleic acids. We present crystal structures of the complex between the SF1B helicase RecD2 from Deinococcus radiodurans and ssDNA in the presence and absence of an ATP analog. These snapshots of the reaction pathway reveal a nucleotide binding-induced conformational change of the two motor domains that is broadly reminiscent of changes observed in other SF1 and SF2 helicases. Together with biochemical data, the structures point to a step size for translocation of one base per ATP hydrolyzed. Moreover, the structures also reveal a mechanism for nucleic acid translocation in the 5'-3' direction by SF1B helicases that is surprisingly different from that of 3'-5' translocation by SF1A enzymes, and explains the molecular basis of directionality.

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DNA Helicase Activity of the RecD Protein from Deinococcus radiodurans

Cited by 38 publications

References 64 publications

Functional Characterization of the Multidomain F Plasmid TraI Relaxase-Helicase

Functional Characterization of the Multidomain F Plasmid TraI Relaxase-Helicase

Kinetics of DNA Unwinding by the RecD2 Helicase from Deinococcus radiodurans

Mechanistic Basis of 5′-3′ Translocation in SF1B Helicases

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