Isolation and characterization of DNA-damage-repair/toleration genes from Arabidopsis thaliana.

摘要

Arabidopsis thaliana was used as a model green plant for studies of DNA damage and repair following UV irradiation. Research results indicated the following: (1) UV (254 nm) fluences that result in measurable cyclobutane pyrimidine dimer (CBPD) levels (about one CBPD per 2 {dollar}times{dollar} 10{dollar}sp4{dollar} nucleotides) have significant physiological effects; (2) photoreactivation is the predominant pathway of UV-induced damage repair in Arabidopsis; (3) Arabidopsis photolyase levels are increased significantly by UV-B irradiation; (4) Arabidopsis photolyase is markedly temperature-sensitive, both in vitro and in vivo.; A genetic technique was developed to isolate Arabidopsis cDNAs encoding putative plant DNA-damage-repair/toleration (DRT) activities by partial complementation of E. coli DNA-repair-deficient mutations. The initial work yielded six unique cDNAs, probably encoding four distinct classes of activities: (1) DRT100 increased resistance of mutant bacteria to UV irradiation, mitomycin C and methyl methanesulfonate (MMS) treatments, supported the growth of {dollar}lambda{lcub}red{rcub}sp-{dollar} {dollar}{lcub}gam{rcub}sp-{dollar} and P1 phages, and promoted conjugational recombination in RecA{dollar}sp-{dollar} bacteria; (2) the activities encoded by DRT101 and DRT102 appear to be UV-specific, since they fail to provide resistance to DNA damage by mitomycin C and MMS; (3) DRT103 conferred UV resistance in a light-dependent manner, but its amino acid sequence shows only marginal similarity to those of microbial photolyases; and (4) DRT111 and DRT112 increased resistance of E.coli resolvase-deficient mutants to UV, mitomycin C, MMS and nitroquinoline oxide, and partially restored the ability of these mutants to carry out conjugal recombination.; The transcript levels of the Arabidopsis DRT genes were examined in response to various DNA-damaging agents. Levels of DRT100 mRNA increased in plants treated with UV-B light, mitomycin C and MMS. The transcript levels of DRT101, whose activity is specific for UV-induced DNA damage in E.coli, became elevated in response not only to UV-B irradiation but also to mitomycin C treatment.