Stimulation of DNA synthesis activity of human DNA polymerase kappa by PCNA.
Humans have three DNA polymerases, Poleta, Polkappa, and Poliota, which are able to promote replication through DNA lesions. However, the mechanism by which these DNA polymerases are targeted to the replication machinery stalled at a lesion site has remained unknown. Here, we provide evidence for the physical interaction of human Polkappa (hPolkappa) with proliferating cell nuclear antigen (PCNA) and show that PCNA, replication factor C (RFC), and replication protein A (RPA) act cooperatively to stimulate the DNA synthesis activity of hPolkappa. The processivity of hPolkappa, however, is not significantly increased in the presence of these protein factors. The efficiency (V(max)/K(m)) of correct nucleotide incorporation by hPolkappa is enhanced approximately 50- to 200-fold in the presence of PCNA, RFC, and RPA, and this increase in efficiency is achieved by a reduction in the apparent K(m) for the nucleotide. Although in the presence of these protein factors, the efficiency of the insertion of an A nucleotide opposite an abasic site is increased approximately 40-fold, this reaction still remains quite inefficient; thus, it is unlikely that hPolkappa would bypass an abasic site by inserting a nucleotide opposite the site.
Haracska
Lajos
L
Sealy Center for Molecular Science, University of Texas Medical Branch, Galveston, Texas 77555-1061, USA.
Unk
Ildiko
I
Johnson
Robert E
RE
Phillips
Barbara B
BB
Hurwitz
Jerard
J
Prakash
Louise
L
Prakash
Satya
S
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D004249:DNA Damage; D004268:DNA-Binding Proteins / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D018809:Proliferating Cell Nuclear Antigen / Q000737:chemistry / Q000378:metabolism*; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011740:Pyrimidine Dimers / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051716:Replication Protein A; D051818:Replication Protein C; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D000099270:Y-Family DNA Polymerases
D004247:DNA; D004268:DNA-Binding Proteins; D004259:DNA-Directed DNA Polymerase; D046911:Macromolecular Substances; D018809:Proliferating Cell Nuclear Antigen; D011506:Proteins; D011740:Pyrimidine Dimers; D011993:Recombinant Fusion Proteins; D051716:Replication Protein A; D051818:Replication Protein C; C497167:RPA1 protein, human; C404302:POLK protein, human; D000099270:Y-Family DNA Polymerases