Replication Protein A Prohibits Diffusion of the PCNA Sliding Clamp along Single-Stranded DNA.
The replicative polymerases cannot accommodate distortions to the native DNA sequence such as modifications (lesions) to the native template bases from exposure to reactive metabolites and environmental mutagens. Consequently, DNA synthesis on an afflicted template abruptly stops upon encountering these lesions, but the replication fork progresses onward, exposing long stretches of the damaged template before eventually stalling. Such arrests may be overcome by translesion DNA synthesis (TLS) in which specialized TLS polymerases bind to the resident proliferating cell nuclear antigen (PCNA) and replicate the damaged DNA. Hence, a critical aspect of TLS is maintaining PCNA at or near a blocked primer/template (P/T) junction upon uncoupling of fork progression from DNA synthesis by the replicative polymerases. The single-stranded DNA (ssDNA) binding protein, replication protein A (RPA), coats the exposed template and might prohibit diffusion of PCNA along the single-stranded DNA adjacent to a blocked P/T junction. However, this idea had yet to be directly tested. We recently developed a unique Cy3-Cy5 Forster resonance energy transfer (FRET) pair that directly reports on the occupancy of DNA by PCNA. In this study, we utilized this FRET pair to directly and continuously monitor the retention of human PCNA at a blocked P/T junction. Results from extensive steady state and pre-steady state FRET assays indicate that RPA binds tightly to the ssDNA adjacent to a blocked P/T junction and restricts PCNA to the upstream duplex region by physically blocking diffusion of PCNA along ssDNA.
Department of Chemistry, The Pennsylvania State University , University Park, Pennsylvania 16802, United States.
Mark
M
Hedglin
Department of Chemistry, The Pennsylvania State University , University Park, Pennsylvania 16802, United States.
Stephen J
0000-0003-3680-3481
SJ
Benkovic
D001483:Base Sequence; D001710:Biotin / Q000737:chemistry; D002232:Carbocyanines / Q000737:chemistry; D002460:Cell Line; D004249:DNA Damage; D004260:DNA Repair; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry; D015870:Gene Expression; D006801:Humans; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000737:chemistry / Q000378:metabolism; D018809:Proliferating Cell Nuclear Antigen / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051716:Replication Protein A / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D002232:Carbocyanines; D004277:DNA, Single-Stranded; D005456:Fluorescent Dyes; D009841:Oligonucleotides; D018809:Proliferating Cell Nuclear Antigen; D011994:Recombinant Proteins; D051716:Replication Protein A; C089187:cyanine dye 3; C085321:cyanine dye 5; D001710:Biotin