SV40 T antigen interactions with ssDNA and replication protein A: a regulatory role of T antigen monomers in lagging strand DNA replication

Nucleic Acids Research
Nichodemus O OnwubikoHeinz P Nasheuer

Abstract

DNA replication is a central process in all living organisms. Polyomavirus DNA replication serves as a model system for eukaryotic DNA replication and has considerably contributed to our understanding of basic replication mechanisms. However, the details of the involved processes are still unclear, in particular regarding lagging strand synthesis. To delineate the complex mechanism of coordination of various cellular proteins binding simultaneously or consecutively to DNA to initiate replication, we investigated single-stranded DNA (ssDNA) interactions by the SV40 large T antigen (Tag). Using single molecule imaging by atomic force microscopy (AFM) combined with biochemical and spectroscopic analyses we reveal independent activity of monomeric and oligomeric Tag in high affinity binding to ssDNA. Depending on ssDNA length, we obtain dissociation constants for Tag-ssDNA interactions (KD values of 10-30 nM) that are in the same order of magnitude as ssDNA binding by human replication protein A (RPA). Furthermore, we observe the formation of RPA-Tag-ssDNA complexes containing hexameric as well as monomeric Tag forms. Importantly, our data clearly show stimulation of primase function in lagging strand Okazaki fragment synthesis by ...Continue Reading

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Citations

Sep 4, 2020·The Journal of Biological Chemistry·Onyekachi E OnonyeMatthew L Bochman
Dec 9, 2020·Antimicrobial Agents and Chemotherapy·Nichodemus O OnwubikoHeinz Peter Nasheuer

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Methods Mentioned

BETA
atomic force microscopy
size exclusion chromatography
electrophoresis
PCR
AFM
Protein Thermal Shift
surface plasmon resonance

Software Mentioned

Igor Pro MFP
GraphPad Prism
GraphPad
Origin Pro
Image J
Protein Thermal Shift
Sedfit
Image SXM
Origin
Image Gauge

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