Abstract
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.
Highlights
Proliferating cell nuclear antigen (PCNA) is a protein that is essential for many DNA metabolic processes including DNA replication, base excision repair, nucleotide excision repair, mismatch repair, and recombination [1,2,3,4]
PCNA is required for translesion synthesis (TLS), which is a pathway for bypassing DNA damage during replication [5,6,7,8,9,10,11,12]
To better understand the role of the subunit interface in causing this TLS block, we have used random mutagenesis to generate a series of PCNA mutants with substitutions in these two strands
Summary
Proliferating cell nuclear antigen (PCNA) is a protein that is essential for many DNA metabolic processes including DNA replication, base excision repair, nucleotide excision repair, mismatch repair, and recombination [1,2,3,4]. PCNA is a ring-shaped homo-trimer that acts as a sliding clamp that encircles DNA forming a scaffold for binding numerous proteins involved in DNA metabolism. Many of these proteins—which include DNA polymerases, mismatch repair proteins, endonucleases, and others—bind PCNA via conserved PCNA-interacting protein (PIP) motifs that dock in a cleft in the front face of the PCNA ring [1,2,4]. During TLS, PCNA is mono-ubiquitylated at Lys-164 in response to replication fork stalling [13]. This is necessary for the switch between the stalled replicative polymerase and a specialized TLS polymerase [14].
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