Identification of New Mutations at the PCNA Subunit Interface that Block Translesion Synthesis.
Proliferating cell nuclear antigen (PCNA) plays an essential role in DNA replication and repair by interacting with a large number of proteins involved in these processes. Two amino acid substitutions in PCNA, both located at the subunit interface, have previously been shown to block translesion synthesis (TLS), a pathway for bypassing DNA damage during replication. To better understand the role of the subunit interface in TLS, we used random mutagenesis to generate a set of 33 PCNA mutants with substitutions at the subunit interface. We assayed the full set of mutants for viability and sensitivity to ultraviolet (UV) radiation. We then selected a subset of 17 mutants and measured their rates of cell growth, spontaneous mutagenesis, and UV-induced mutagenesis. All except three of these 17 mutants were partially or completely defective in induced mutagenesis, which indicates a partial or complete loss of TLS. These results demonstrate that the integrity of the subunit interface of PCNA is essential for efficient TLS and that even conservative substitutions have the potential to disrupt this process.
Department of Biochemistry, Carver College of Medicine, University of Iowa, Iowa City, Iowa, 52242, United States of America.
Christine M
CM
Kondratick
Department of Biochemistry, Carver College of Medicine, University of Iowa, Iowa City, Iowa, 52242, United States of America.
Elizabeth M
EM
Boehm
Department of Chemistry, Creighton University, Omaha, Nebraska, 68178, United States of America.
Lynne M
LM
Dieckman
Department of Biochemistry, Carver College of Medicine, University of Iowa, Iowa City, Iowa, 52242, United States of America.
Kyle T
KT
Powers
Department of Biochemistry, Carver College of Medicine, University of Iowa, Iowa City, Iowa, 52242, United States of America.
Julio C
JC
Sanchez
Department of Biochemistry, Carver College of Medicine, University of Iowa, Iowa City, Iowa, 52242, United States of America.
Samuel R
SR
Mueting
Department of Biochemistry, Carver College of Medicine, University of Iowa, Iowa City, Iowa, 52242, United States of America.
M Todd
MT
Washington
D004249:DNA Damage / Q000235:genetics; D004261:DNA Replication / Q000235:genetics; D016296:Mutagenesis; D009154:Mutation / Q000235:genetics*; D018809:Proliferating Cell Nuclear Antigen / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014466:Ultraviolet Rays
D018809:Proliferating Cell Nuclear Antigen; D029701:Saccharomyces cerevisiae Proteins