[Construction of the recQ double mutants and analysis of adversity in Deinococcus radiodurans].
As a subfamily member of SF1 superfamily, the RecO helicases are highly conserved in evolution and are required for maintaining genome stability in all organisms. Loss of RecO helicase function leads to a breakdown in the maintenance of genome integrity, in particular hyper-recombination. Named after the recQ gene of Escherichia coli, lower eukaryotic species generally only contain a single RecQ family representative; for example, Sgsl in the budding yeast, Saccharomyces cerevisiae, and Rqhl in the fission yeast, Schizosaccharomyces pombe. There are, however, multiple members in most higher organisms, with five being present in humans. Defects in three of these human RecQ helicases give rise to defined clinical disorders associated with cancer predisposition and variable aspects of premature aging. Deinococcus radiodurans encodes two recQ genes with unusual domain: DR1289 and DR2444, whose functions, however, remain obscure currently. DR1289 contains three tandem copies of the C-terminal helicase-RNase D (HRDC) domain, instead of the single copy present in all other bacteria except Neisseria that similarly possesses three copies. DR2444 contains a HRDC domain and a domain homologous to cystathionine gamma-lyase; this is the first example of an HRDC domain that is not associated with either a helicase or a nuclease. In this study, a fusion DNA fragment carrying kanamycin resistance gene with the D. radiodurans groEL promoter, chloramphenicol resistance gene with KAT promoter was cloned by PCR amplification and reversely inserted into the recQ locus in the genome of the wild-type strain RI. Three resulting recQ-deficient strains, designated deltaDR1289, deltaDR2444 and deltarecQ (double mutation), were constructed. Results show that deltaDR1289 and delta recQ were very sensitive to ionizing radiation and H2O2, while delta DR2444 and wild strain R1 were not. The phenotype of delta DR1289 was similar to many RecQ helicase mutants. Therefore, it was presumed that DR1289 was the necessary gene in maintaining the extreme resistance to DNA damaging agents, whereas DR2444 was not. Further research based on genetic and biochemical approaches should help to gain a better understanding of the genes involved in DNA repair.
Huang
Li-Fen
LF
Institute of Nuclear-Agricultral Sciences, Zhejiang University, Hangzhou 310029, China. h_lf@sina.com.cn
Zhang
Shao-Wen
SW
Hua
Xiao-Ting
XT
Gao
Guan-Jun
GJ
Hua
Yue-Jin
YJ
D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D034301:Deinococcus / Q000201:enzymology / Q000235:genetics* / Q000528:radiation effects*; D005720:Gamma Rays; D009154:Mutation; D053484:RecQ Helicases / Q000235:genetics* / Q000378:metabolism
D001426:Bacterial Proteins; D053484:RecQ Helicases