Ubiquitylation of proliferating cell nuclear antigen and recruitment of human DNA polymerase eta.
This study investigated the requirement for ubiquitylation of PCNA at lysine 164 during polymerase eta-dependent translesion synthesis (TLS) of site-specific cis-syn cyclobutane thymine dimers (T (wedge)T). The in vitro assay recapitulated origin-dependent initiation, fork assembly, and semiconservative, bidirectional replication of double-stranded circular DNA substrates. A phosphocellulose column was used to fractionate HeLa cell extracts into two fractions; flow-through column fraction I (CFI) contained endogenous PCNA, RPA, ubiquitin-activating enzyme E1, and ubiquitin conjugase Rad6, and eluted column fraction II (CFII) included pol delta, pol eta, and RFC. CFII supplemented with purified recombinant RPA and PCNA (wild type or K164R, in which lysine was replaced with arginine) was competent for DNA replication and TLS. K164R-PCNA complemented CFII for these activities to the same extent and efficiency as wild-type PCNA. CFII mixed with CFI (endogenous PCNA, E1, Rad6) exhibited enhanced DNA replication activity, but the same TLS efficiency determined with the purified proteins. These results demonstrate that PCNA ubiquitylation at K164 of PCNA is not required in vitro for pol eta to gain access to replication complexes at forks stalled by T (wedge)T and to catalyze TLS across this dimer.
Nikolaishvili-Feinberg
Nana
N
Department of Pathology and Laboratory Medicine, Lineberger Comprehensive Cancer Center and Center for Environmental Health and Susceptibility, University of North Carolina, Chapel Hill, North Carolina 27599, USA.
Jenkins
G Scott
GS
Nevis
Kathleen R
KR
Staus
Dean P
DP
Scarlett
Cameron O
CO
Unsal-Kaçmaz
Keziban
K
Kaufmann
William K
WK
Cordeiro-Stone
Marila
M
D001483:Base Sequence; D017931:DNA Primers; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D019281:Dimerization; D004587:Electrophoresis, Agar Gel / Q000379:methods; D006367:HeLa Cells; D006801:Humans; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism*; D013936:Thymidine / Q000378:metabolism; D025801:Ubiquitin / Q000378:metabolism*; D000099270:Y-Family DNA Polymerases
D017931:DNA Primers; D004259:DNA-Directed DNA Polymerase; D018809:Proliferating Cell Nuclear Antigen; D013936:Thymidine; D025801:Ubiquitin; C112338:Rad30 protein; D000099270:Y-Family DNA Polymerases