Arabidopsis thaliana Y-family DNA polymerase eta catalyses translesion synthesis and interacts functionally with PCNA2.
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.
Anderson
Heather J
HJ
School of Life and Environmental Sciences, Deakin University, Geelong, Vic. 3217, Australia.
Vonarx
Edward J
EJ
Pastushok
Landon
L
Nakagawa
Mayu
M
Katafuchi
Atsushi
A
Gruz
Petr
P
Di Rubbo
Antonio
A
Grice
Desma M
DM
Osmond
Megan J
MJ
Sakamoto
Ayako N
AN
Nohmi
Takehiko
T
Xiao
Wei
W
Kunz
Bernard A
BA
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000235:genetics* / Q000378:metabolism / Q000528:radiation effects; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D004249:DNA Damage; D004260:DNA Repair; D004261:DNA Replication; D004269:DNA, Bacterial / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism*; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009154:Mutation; D018517:Plant Roots / Q000235:genetics / Q000378:metabolism / Q000528:radiation effects; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism*; D018749:RNA, Plant / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D020798:Two-Hybrid System Techniques; D014466:Ultraviolet Rays; D000099270:Y-Family DNA Polymerases
D029681:Arabidopsis Proteins; D004269:DNA, Bacterial; D018076:DNA, Complementary; D004259:DNA-Directed DNA Polymerase; D018809:Proliferating Cell Nuclear Antigen; D018749:RNA, Plant; D011994:Recombinant Proteins; C036483:T-DNA; C112338:Rad30 protein; D000099270:Y-Family DNA Polymerases