Identification of two functional PCNA-binding domains in human DNA polymerase κ.
Previously, we have shown that human DNA polymerase (Pol) η has two functional PCNA-binding motifs, PIP1 and PIP2, and that a C-terminal deletion of Polη that lacks the ubiquitin-binding UBZ domain and the PIP2 domain but retains the PIP1 domain promotes normal levels of translesion synthesis (TLS) opposite a cis-syn TT dimer in human cells. Here, we identify two PIP domains in Polκ and show that TLS occurs normally in human fibroblast cells in which the pip1 or pip2 mutant Polκ is expressed, but mutational inactivation of both PIP domains renders Polκ nonfunctional in TLS opposite the thymine glycol lesion. Thus, the two PIP domains of Polκ function redundantly in TLS opposite this DNA lesion in human cells. However, and surprisingly, whereas mutational inactivation of the PIP1 domain completely inhibits the stimulation of DNA synthesis by Polκ in the presence of proliferating cell nuclear antigen (PCNA), replication factor C, and replication protein A, mutations in PIP2 have no adverse effect on PCNA-dependent DNA synthesis. This raises the possibility that activation of Polκ PIP2 as a PCNA-binding domain occurs during TLS in human cells and that protein-protein interactions and post-transcriptional modifications are involved in such activation.
Department of Biochemistry and Molecular Biology, University of Texas Medical Branch at Galveston, 301 University Blvd., Galveston, Texas, 77555-1061, USA.
Jung-Hoon
JH
Yoon
Narottam
N
Acharya
Jeseong
J
Park
Debashree
D
Basu
Satya
S
Prakash
Louise
L
Prakash
D002352:Carrier Proteins / Q000378:metabolism*; D002460:Cell Line; D004247:DNA / Q000378:metabolism*; D004261:DNA Replication / Q000187:drug effects; D004268:DNA-Binding Proteins; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D009154:Mutation; D054730:Protein Interaction Domains and Motifs; D051716:Replication Protein A / Q000378:metabolism; D051818:Replication Protein C / Q000378:metabolism; D000099270:Y-Family DNA Polymerases
D002352:Carrier Proteins; D004247:DNA; D004268:DNA-Binding Proteins; D004259:DNA-Directed DNA Polymerase; D051716:Replication Protein A; D051818:Replication Protein C; C425208:PCLAF protein, human; C404302:POLK protein, human; D000099270:Y-Family DNA Polymerases