Involvement of AtPolλ in repair of high salt and DNA cross linking agent induced double strand breaks in Arabidopsis thaliana.

DNA polymerase λ (Pol λ) is the sole member of family X DNA polymerase in plants and plays crucial role in nuclear DNA damage repair. Here we report transcriptional up-regulation of Arabidopsis thaliana DNA polymerase λ (AtPolλ) in response to abiotic and genotoxic stress including salinity and DNA cross-linking agent mitomycin C (MMC). The increased sensitivity of atpolλ knockout mutants towards high salinity and MMC treatment with high levels of accumulation of double strand breaks (DSBs) than wild-type plants and delayed repair of DSBs have suggested requirement of Pol λ in DSB repair in plants. AtPolλ overexpression moderately complemented the deficiency of DSB repair capacity in atpolλ mutants. Transcriptional up-regulation of major non-homologous end joining (NHEJ) pathway genes KU80, X-RAY CROSS COMPLEMENTATION PROTEIN4 (XRCC4) and DNA ligase 4 (Lig4) along with AtPolλ in Arabidopsis seedlings and the increased sensitivity of atpolλ-2/atxrcc4 and atpolλ-2/atlig4 double mutants towards high salinity and MMC treatments indicated involvement of NHEJ mediated repair of salinity and MMC induced DSBs. The suppressed expression of NHEJ genes in atpolλ mutants suggested complex transcriptional regulation of NHEJ genes. Pol λ interacted directly with XRCC4 and Lig4 via its N-terminal BRCT domain in yeast two hybrid system, while increased sensitivity of BRCT deficient Pol λ expressing transgenic atpolλ-2 mutants towards genotoxins indicated importance of BRCT domain of AtPolλ in mediating the interactions for processing DSBs. Our findings provide evidences for direct involvement of DNA Pol λ in repair of DSBs in plant genome.