SUMMARY: Upon blockage of chromosomal replication by DNA lesions, Y-family polymerases interact with monoubiquitylated proliferating cell nuclear antigen (PCNA) to catalyse translesion synthesis (TLS) and restore replication fork progression. Here, we assessed the roles of Arabidopsis thaliana POLH, which encodes a homologue of Y-family polymerase eta (Poleta), PCNA1 and PCNA2 in TLS-mediated UV resistance. A T-DNA insertion in POLH sensitized the growth of roots and whole plants to UV radiation, indicating that AtPoleta contributes to UV resistance. POLH alone did not complement the UV sensitivity conferred by deletion of yeast RAD30, which encodes Poleta, although AtPoleta exhibited cyclobutane dimer bypass activity in vitro, and interacted with yeast PCNA, as well as with Arabidopsis PCNA1 and PCNA2. Co-expression of POLH and PCNA2, but not PCNA1, restored normal UV resistance and mutation kinetics in the rad30 mutant. A single residue difference at site 201, which lies adjacent to the residue (lysine 164) ubiquitylated in PCNA, appeared responsible for the inability of PCNA1 to function with AtPoleta in UV-treated yeast. PCNA-interacting protein boxes and an ubiquitin-binding motif in AtPoleta were found to be required for the restoration of UV resistance in the rad30 mutant by POLH and PCNA2. These observations indicate that AtPoleta can catalyse TLS past UV-induced DNA damage, and links the biological activity of AtPoleta in UV-irradiated cells to PCNA2 and PCNA- and ubiquitin-binding motifs in AtPoleta.
Mutational Analysis, Nucleotide Analogs / Template Lesions, Accessory Proteins/Complexes, Nucleotide Incorporation, Alignments, Source / Purification