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Identification of two functional PCNA ‐binding domains in human DNA polymerase κ
Author(s) -
Yoon JungHoon,
Acharya Narottam,
Park Jeseong,
Basu Debashree,
Prakash Satya,
Prakash Louise
Publication year - 2014
Publication title -
genes to cells
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.912
H-Index - 115
eISSN - 1365-2443
pISSN - 1356-9597
DOI - 10.1111/gtc.12156
Subject(s) - proliferating cell nuclear antigen , biology , dna replication , dna polymerase , microbiology and biotechnology , processivity , dna , dna clamp , biochemistry , gene , rna , reverse transcriptase
Previously, we have shown that human DNA polymerase (Pol) η has two functional PCNA ‐binding motifs, PIP 1 and PIP 2, and that a C‐terminal deletion of Polη that lacks the ubiquitin‐binding UBZ domain and the PIP 2 domain but retains the PIP 1 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 PIP 1 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 PIP 2 have no adverse effect on PCNA ‐dependent DNA synthesis. This raises the possibility that activation of Polκ PIP 2 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.