Arabidopsis thaliana AtPOLK encodes a DinB-like DNA polymerase that extends mispaired primer termini and is highly expressed in a variety of tissues.
Cell survival after DNA damage depends on specialized DNA polymerases able to perform DNA synthesis on imperfect templates. Most of these enzymes belong to the recently discovered Y-family of DNA polymerases, none of which has been previously described in plants. We report here the isolation, functional characterization and expression analysis of a plant representative of the Y-family. This polymerase, which we have termed AtPolkappa, is a homolog of Escherichia coli pol IV and human pol kappa, and thus belongs to the DinB subfamily. We purified AtPolkappa and found a template-directed DNA polymerase, endowed with limited processivity that is able to extend primer-terminal mispairs. The activity and processivity of AtPolkappa are enhanced markedly upon deletion of 193 amino acids (aa) from its carboxy (C)-terminal domain. Loss of this region also affects the nucleotide selectivity of the enzyme, leading to the incorporation of both dCTP and dTTP opposite A in the template. We detected three cDNA forms, which result from the alternative splicing of AtPOLK mRNA and have distinct patterns of expression in different plant organs. Histochemical localization of beta-glucuronidase (GUS) activity in transgenic plants revealed that the AtPOLK promoter is active in endoreduplicating cells, suggesting a possible role during consecutive DNA replication cycles in the absence of mitosis.
García-Ortiz
Maria Victoria
MV
Departamento de Genética, Universidad de Córdoba, 14071 Córdoba, Spain.
Ariza
Rafael R
RR
Hoffman
Peter D
PD
Hays
John B
JB
Roldán-Arjona
Teresa
T
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000235:genetics*; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000235:genetics*; D015870:Gene Expression; D015723:Gene Library; D008969:Molecular Sequence Data; D010802:Phylogeny; D030821:Plants, Genetically Modified; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D000099270:Y-Family DNA Polymerases
D029681:Arabidopsis Proteins; D004259:DNA-Directed DNA Polymerase; D011993:Recombinant Fusion Proteins; C498615:POLK protein, Arabidopsis; D000099270:Y-Family DNA Polymerases