Dataset Information

Effects of Conserved Wedge Domain Residues on DNA Binding Activity of Deinococcus radiodurans RecG Helicase.

ABSTRACT: Deinococcus radiodurans is extremely resistant to ionizing radiation and has an exceptional ability to repair DNA damage caused by various DNA-damaging agents. D. radiodurans uses the same DNA-repair strategies as other prokaryotes, but certain proteins involved in the classical DNA repair machinery have characteristics different from their counterparts. RecG helicase, which unwinds a variety of branched DNA molecules, such as Holliday junctions (HJ) and D-loops, plays important roles in DNA repair, recombination, and replication. Primary sequence analysis of RecG from a number of bacterial species revealed that three amino acids (QPW) in the DNA-binding wedge domain (WD) are well-conserved across the Deinococcus RecG proteins. Interactions involving these conserved residues and DNA substrates were predicted in modeled domain structures of D. radiodurans RecG (DrRecG). Compared to the WD of Escherichia coli RecG protein (EcRecG) containing FSA amino acids corresponding to QPW in DrRecG, the HJ binding activity of DrRecG-WD was higher than that of EcRecG-WD. Reciprocal substitution of FSA and QPW increased and decreased the HJ binding activity of the mutant WDs, EcRecG-WD_QPW, and DrRecG-WD_FSA, respectively. Following ?-irradiation treatment, the reduced survival rate of DrRecG mutants (?recG) was fully restored by the expression of DrRecG, but not by that of EcRecG. EcRecG_QPW also enhanced ?-radioresistance of ?recG, whereas DrRecG_FSA did not. ?recG cells complemented in trans by DrRecG and EcRecG_QPW reconstituted an intact genome within 3 h post-irradiation, as did the wild-type strain, but ?recG with EcRecG and DrRecG_FSA exhibited a delay in assembly of chromosomal fragments induced by ?-irradiation. These results suggested that the QPW residues facilitate the association of DrRecG with DNA junctions, thereby enhancing the DNA repair efficiency of DrRecG.

SUBMITTER: Jeong SW

PROVIDER: S-EPMC7889586 | biostudies-literature | 2021

REPOSITORIES: biostudies-literature

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Effects of Conserved Wedge Domain Residues on DNA Binding Activity of Deinococcus radiodurans RecG Helicase.

Jeong Sun-Wook SW Kim Min-Kyu MK Zhao Lei L Yang Seul-Ki SK Jung Jong-Hyun JH Lim Heon-Man HM Lim Sangyong S

Frontiers in genetics 20210204

Deinococcus radiodurans is extremely resistant to ionizing radiation and has an exceptional ability to repair DNA damage caused by various DNA-damaging agents. D. radiodurans uses the same DNA-repair strategies as other prokaryotes, but certain proteins involved in the classical DNA repair machinery have characteristics different from their counterparts. RecG helicase, which unwinds a variety of branched DNA molecules, such as Holliday junctions (HJ) and D-loops, plays important ro ...[more]

PMID: 33613647

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Project description:Deinococcus radiodurans R1 is one of the most radiation-resistant organisms known and is able to repair an unusually large amount of DNA damage without induced mutation. Single-stranded DNA-binding (SSB) protein is an essential protein in all organisms and is involved in DNA replication, recombination and repair. The published genomic sequence from Deinococcus radiodurans includes a putative single-stranded DNA-binding protein gene (ssb; DR0100) requiring a translational frameshift for synthesis of a complete SSB protein. The apparently tripartite gene has inspired considerable speculation in the literature about potentially novel frameshifting or RNA editing mechanisms. Immediately upstream of the ssb gene is another gene (DR0099) given an ssb-like annotation, but left unexplored.A segment of the Deinococcus radiodurans strain R1 genome encompassing the ssb gene has been re-sequenced, and two errors involving omitted guanine nucleotides have been documented. The corrected sequence incorporates both of the open reading frames designated DR0099 and DR0100 into one contiguous ssb open reading frame (ORF). The corrected gene requires no translational frameshifts and contains two predicted oligonucleotide/oligosaccharide-binding (OB) folds. The protein has been purified and its sequence is closely related to the Thermus thermophilus and Thermus aquaticus SSB proteins. Like the Thermus SSB proteins, the SSBDr functions as a homodimer. The Deinococcus radiodurans SSB homodimer stimulates Deinococcus radiodurans RecA protein and Escherichia coli RecA protein-promoted DNA three-strand exchange reactions with at least the same efficiency as the Escherichia coli SSB homotetramer.The correct Deinococcus radiodurans ssb gene is a contiguous open reading frame that codes for the largest bacterial SSB monomer identified to date. The Deinococcus radiodurans SSB protein includes two OB folds per monomer and functions as a homodimer. The Deinococcus radiodurans SSB protein efficiently stimulates Deinococcus radiodurans RecA and also Escherichia coli RecA protein-promoted DNA strand exchange reactions. The identification and purification of Deinococcus radiodurans SSB protein not only allows for greater understanding of the SSB protein family but provides an essential yet previously missing player in the current efforts to understand the extraordinary DNA repair capacity of Deinococcus radiodurans.

Dataset Information

Effects of Conserved Wedge Domain Residues on DNA Binding Activity of Deinococcus radiodurans RecG Helicase.

Publications

Effects of Conserved Wedge Domain Residues on DNA Binding Activity of <i>Deinococcus radiodurans</i> RecG Helicase.

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