Functional Analysis Of CSAat1A And CSAat1B Proteins In Arabidopsis

Increasing amounts of UV-B radiation reach the earth's surface each year due to a reduction in the stratospheric ozone layer; these increased levels of radiation have significant effects on plant growth and development. Through forward genetic screening, we identified a UV-sensitive mutant (csaat1a-3), in which expression of CSAat1A, a Cockayne Syndrome A-like gene, is reduced due to insertion of a T-DNA in the promoter region. Arabidopsis thaliana lacking CSAatlA or its homolog CSAat1B is more sensitive to UV-B and the genotoxic drug Methyl Methanesulfonate and exhibits reduced transcription-coupled repair activity. ELISA results indicate that wild type and the mutant had similar UV-B based DNA damage and that the DNA damage repair machinery is impaired in the csaat1a and csaat1b mutants.Yeast two-hybrid analysis indicated that both CSAat1A and CSAat1B (CSAat1A and B) in-teract with DDB1A (UV-Damage DNA Binding Protein 1). Co-immunoprecipitation assays demonstrated that CSAat1A and B associate with the CUL4 (CULLIN4)-DDB1A complex in Arabidopsis. A split-YFP assay showed that this in-teraction occurs in the nucleus, consistent with the idea that the CUL4-DDB1-CSA complex functions as a nuclear E3 ubiquitin ligase. CSAat1A and CSAat1B formed heterotetramers in Arabidopsis. Taken together, our data indicate that the plant CUL4-DDB1CSAat1A and B complex represents a unique mechanism to promote ubiquit-ination of substrates in response to DNA damage.