PCNA tool belts and polymerase bridges form during translesion synthesis.
Large multi-protein complexes play important roles in many biological processes, including DNA replication and repair, transcription, and signal transduction. One of the challenges in studying such complexes is to understand their mechanisms of assembly and disassembly and their architectures. Using single-molecule total internal reflection (TIRF) microscopy, we have examined the assembly and disassembly of the multi-protein complex that carries out translesion synthesis, the error-prone replication of damaged DNA. We show that the ternary complexes containing proliferating cell nuclear antigen (PCNA) and two non-classical DNA polymerases, Rev1 and DNA polymerase η, have two architectures: PCNA tool belts and Rev1 bridges. Moreover, these complexes are dynamic and their architectures can interconvert without dissociation. The formation of PCNA tool belts and Rev1 bridges and the ability of these complexes to change architectures are likely means of facilitating selection of the appropriate non-classical polymerase and polymerase-switching events.
Department of Biochemistry, Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA.
Elizabeth M
EM
Boehm
Department of Biochemistry, Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA.
Maria
M
Spies
Department of Biochemistry, Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA todd-washington@uiowa.edu.
M Todd
MT
Washington
D020816:Amino Acid Motifs; D019943:Amino Acid Substitution; D001665:Binding Sites; D004247:DNA / Q000096:biosynthesis*; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000378:metabolism*; D005453:Fluorescence; D007700:Kinetics; D046912:Multiprotein Complexes / Q000378:metabolism; D050505:Mutant Proteins / Q000378:metabolism; D009713:Nucleotidyltransferases / Q000737:chemistry / Q000378:metabolism*; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism*; D011485:Protein Binding; D000072417:Protein Domains; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism*; D000099270:Y-Family DNA Polymerases
D004247:DNA; D004259:DNA-Directed DNA Polymerase; D046912:Multiprotein Complexes; D050505:Mutant Proteins; D009713:Nucleotidyltransferases; D018809:Proliferating Cell Nuclear Antigen; D029701:Saccharomyces cerevisiae Proteins; C100951:REV1 protein, S cerevisiae; D000099270:Y-Family DNA Polymerases; C112338:Rad30 protein