AtPolλ, A Homolog of Mammalian DNA Polymerase λ in Arabidopsis thaliana, is Involved in the Repair of UV-B Induced DNA Damage Through the Dark Repair Pathway

Abstract

Plants are constantly exposed to a wide range of environmental genotoxic stress factors including obligatory exposure to UV radiation in sunlight. Here, we report the functional characterization of a DNA repair protein, AtPolλ, a homolog of mammalian DNA polymerase λ in Arabidopsis, in relation to its role in repair of UV-B-induced DNA damage during early stages of seedling development. The abundance of the AtPolλ transcript and the protein levels were distinctly increased in response to UV-B irradiation in 6-day-old wild-type seedlings. Growth of atpolλ mutant seedlings, deficient in AtPolλ expression, was more sensitive to UV-B radiation compared with wild-type plants when seeds were exposed to UV-B radiation before germination. The atpolλ mutants showed accumulation of relatively higher amounts of DNA lesions than wild-type plants following UV-B exposure and were less proficient in repair of UV-induced DNA damage. Increased accumulation of AtPolλ protein in UV-B-irradiated 6-day-old wild-type seedlings during the dark recovery period has indicated a possible role for the protein in repair of UV-B-induced lesions in the dark. Overexpression of AtPolλ in the atpolλ mutant line partially complemented the repair proficiency of UV-B-induced DNA damage. In vitro repair synthesis assays using whole-cell extracts from the wild-type and atpolλ mutant line have further demonstrated the role of AtPolλ in repair synthesis of UV-B-damaged DNA in the dark through an excision repair mechanism. Overall, our results have indicated the possible involvement of AtPolλ in a plant's response for repair of UV-B-mediated DNA damage during seedling development.

Involvement of AtPolλ in the Repair of High Salt- and DNA Cross-Linking Agent-Induced Double Strand Breaks in Arabidopsis

“…AtPoll (AT1G10520) belongs to family X DNA Pol based on its sequence homology with mammalian DNA Pol b, DNA Pol m, and terminal deoxynucleotidyl transferase and appears to be the single representative of family X DNA Pols in plants to carry out nuclear DNA repair functions (Amoroso et al, 2011;Roy et al, 2011). The full-length AtPoll, expressed in Escherichia coli cells, has been found as a single polypeptide protein with an intrinsic DNA polymerase activity (Supplemental Fig.…”

“…Recent studies have demonstrated roles of DNA Pol l in UV-B-induced DNA damage repair and translesion DNA synthesis in the repair of oxidative DNA damage (Amoroso et al, 2011;Roy et al, 2011), suggesting the requirement of Pol l in different repair pathways in plants. To further characterize the transcriptional induction of the Pol l gene in response to abiotic and genotoxic agent treatments, we next examined AtPoll message levels in 7-d-old wild-type Arabidopsis seedlings subjected to treatment with various stress factors, as described in "Materials and Methods."…”

“…Plants were transformed by Agrobacteriummediated floral dip method. Wild-type Col-0 and atpoll-1 and atpoll-2 transgenic lines overexpressing the 1.59-kb full-length cDNA of AtPoll under the control of the CaMV 35S constitutive promoter were described previously (Roy et al, 2011). Arabidopsis transgenic lines containing the 789-bp upstream promoter sequence of AtPoll in fusion with the GUS reporter have been described previously (Roy et al, 2012).…”

“…Total protein extracts were prepared from approximately 100 mg of plant tissues by following the method described previously (Roy et al, 2011). Protein concentration in each sample was determined by the Bradford assay (Bradford, 1976) using bovine serum albumin as a standard (Fraction V; Sigma).…”

“…Recently, we have reported the requirement of Arabidopsis (Arabidopsis thaliana) DNA Pol l (AtPoll) in the repair of UV-B-induced DNA damage via the nucleotide excision repair pathway (Roy et al, 2011). In addition, the role of DNA Pol l in high-fidelity translesion DNA synthesis in response to oxidative DNA damage has been demonstrated in Arabidopsis (Amoroso et al, 2011).…”