RT Journal Article
T1 Replicative DNA polymerases promote active displacement of SSB proteins during lagging strand synthesis
A1 Cerrón, Fernando
A1 Lorenzo, Sara de
A1 Lemishko, Kateryna M.
A1 Ciesielski, Grzegorz L.
A1 Kaguni, Laurie S.
A1 Cao García, Francisco Javier
A1 Ibarra, Borja
AB Genome replication induces the generation of large stretches of single-stranded DNA (ssDNA) intermediates that are rapidly protected by single-stranded DNA-binding (SSB) proteins. To date, the mechanism by which tightly bound SSBs are removed from ssDNA by the lagging strand DNA polymerase without compromising the advance of the replication fork remains unresolved. Here, we aimed to address this question by measuring, with optical tweezers, the real-time replication kinetics of the human mitochondrial and bacteriophage T7 DNA polymerases on free-ssDNA, in comparison with ssDNA covered with homologous and non-homologous SSBs under mechanical tension. We find important differences between the force dependencies of the instantaneous replication rates of each polymerase on different substrates. Modeling of the data supports a mechanism in which strong, specific polymerase-SSB interactions, up to similar to 12 k(B) T, are required for the polymerase to dislodge SSB from the template without compromising its instantaneous replication rate, even under stress conditions that may affect SSB-DNA organization and/or polymerase-SSB communication. Upon interaction, the elimination of template secondary structure by SSB binding facilitates the maximum replication rate of the lagging strand polymerase. In contrast, in the absence of polymerase-SSB interactions, SSB poses an effective barrier for the advance of the polymerase, slowing down DNA synthesis.
PB Oxford University Press
SN 0305-1048
YR 2019
FD 2019-06-20
LK https://hdl.handle.net/20.500.14352/13787
UL https://hdl.handle.net/20.500.14352/13787
LA eng
NO © The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research. We are grateful to Dr J. Jarillo for helping with data analysis and to members of B. Ibarra lab for useful discussions. Spanish Ministry of Economy and Competitiveness [FIS2015–67765-R to F.J.C., BFU2012–31825, BFU2015–63714-R to B.I.]; National Institutes of Health [GM45925 to L.S.K.]; Comunidad de Madrid [NanoMagCOST P2018 INMT-4321]; Programa de Financiación Universidad Complutense de Madrid–Santander Universidades [CT45/15-CT46/15 to F.C.]; Ministerio de Educación Cultura y Deporte [FPU2014/06867 to K.M.L.]; IMDEA Nanociencia acknowledges support from the ‘Severo Ochoa’ Programme for Centers of Excellence in R&D [MINECO, Grant SEV-2016–0686]. Funding for open access charge: Spanish Ministry of Economy and Competitiveness [BFU2015-63714-R].
NO Ministerio de Economía y Competitividad (MINECO)
NO Comunidad de Madrid
NO Universidad Complutense de Madrid/Banco de Santander
NO Centro de Excelencia Severo Ochoa
NO National Institutes of Health United States Department of Health & Human Services National Institutes of Health (NIH) - USA
NO Ministerio de Educación, Cultura y Deporte
DS Docta Complutense
RD 10 abr 2025