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

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...

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Bibliographic Details
Published in:	Nucleic acids research Vol. 48; no. 7; pp. 3657 - 3677
Main Authors:	Onwubiko, Nichodemus O, Borst, Angela, Diaz, Suraya A, Passkowski, Katharina, Scheffel, Felicia, Tessmer, Ingrid, Nasheuer, Heinz P
Format:	Journal Article
Language:	English
Published:	England Oxford University Press 17-04-2020
Subjects:	Genome Integrity, Repair and
Online Access:	Get full text
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Summary:	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 monomeric Tag whereas hexameric Tag inhibits the reaction, redefining DNA replication initiation on the lagging strand.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 The authors wish it to be known that, in their opinion, the first five authors should be regarded as Joint First Authors.
ISSN:	0305-1048 1362-4962
DOI:	10.1093/nar/gkaa138