A UVB‐hypersensitive mutant in Arabidopsis thaliana is defective in the DNA damage response

Abstract

SUMMARYTo investigate UVB DNA damage response in higher plants, we used a genetic screen to isolate Arabidopsis thaliana mutants that are hypersensitive to UVB irradiation, and isolated a UVB-sensitive mutant, termed suv2 (for sensitive to UV 2) that also displayed hypersensitivity to c-radiation and hydroxyurea. This phenotype is reminiscent of the Arabidopsis DNA damage-response mutant atr. The suv2 mutation was mapped to the bottom of chromosome 5, and contains an insertion in an unknown gene annotated as MRA19.1. RT-PCR analysis with specific primers to MRA19.1 detected a transcript consisting of 12 exons. The transcript is predicted to encode a 646 amino acid protein that contains a coiled-coil domain and two instances of predicted PIKK target sequences within the N-terminal region. Fusion proteins consisting of the predicted MRA19.1 and DNA-binding or activation domain of yeast transcription factor GAL4 interacted with each other in a yeast twohybrid system, suggesting that the proteins form a homodimer. Expression of CYCB1;1:GUS gene, which encodes a labile cyclin:GUS fusion protein to monitor mitotic activity by GUS activity, was weaker in the suv2 plant after c-irradiation than in the wild-type plants and was similar to that in the atr plants, suggesting that the suv2 mutant is defective in cell-cycle arrest in response to DNA damage. Overall, these results suggest that the gene disrupted in the suv2 mutant encodes an Arabidopsis homologue of the ATR-interacting protein ATRIP.

RAD5a and REV3 function in two alternative pathways of DNA-damage tolerance in Arabidopsis

“…The rad5a-3 rev3-2 homozygous double mutants were generated through crossing rad5a-3 and rev3-2 plants. Characterization of the same rev3-2 allele has been previously reported (Sakamoto et al, 2003). On the other hand, although two other RAD5A T-DNA insertion lines SALK_124891 (rad5a-1) and SALK_047150 (rad5a-2) have been reported (Chen et al, 2008), the rad5-3 line utilized in this study has not been previously characterized.…”

“…Specifically relevant to this study, Arabidopsis is an excellent multicellular model organism to study genes involved in genome stability because it is more tolerant to genome instability than mammalian models such as mice. In mice, embryonic lethality has been reported in many cases when genes related to DNA repair are deleted (Wu et al, 2009 Ries et al, 2000;Sakamoto et al, 2003). It appears that major pathways known in yeast and/or animals including photoreactivation, nucleotide excision repair, and DDT are conserved in plants (Britt, 1999;Hays, 2002).…”

“…In the TLS branch, genes encoding Rev3 (Sakamoto et al, 2003), Rev7, Rev1 (Takahashi et al, 2005), and Polη (Anderson et al, 2008;Curtis and Hays, 2007) have been reported (Table 1.1). Inactivation of these genes causes various extents of sensitivity to UV as well as other DNA-damaging agents.…”

“…were obtained from the Arabidopsis Biological Resource Center (ABRC, www.arabidopsis.org), and the alleles were designated rad5a-3 in this study and rev3-2 in a previous report (Sakamoto et al, 2003), respectively. The rad5a-3 rev3-2 homozygous double mutants were generated through crossing rad5a-3 and rev3-2 plants.…”

“…It has been reported that rad5a and rev3 single mutants show no morphological changes under normal growth conditions compared to the wild-type plants; however, they display characteristic sensitivity to DNA-damaging agents (Chen et al, 2008;Sakamoto et al, 2003).…”