Replicative DNA polymerases promote active displacement of SSB proteins during lagging strand synthesis

Authors

Fernando Cerrón, Instituto Madrileño de Estudios Avanzados en Nanociencia, IMDEA Nanociencia. 28049 Madrid, Spain.
Sara de Lorenzo, Instituto Madrileño de Estudios Avanzados en Nanociencia, IMDEA Nanociencia. 28049 Madrid, Spain.
Kateryna M. Lemishko, Instituto Madrileño de Estudios Avanzados en Nanociencia, IMDEA Nanociencia. 28049 Madrid, Spain.
Grzegorz L. Ciesielski, Department of Biochemistry and Molecular Biology and Center for Mitochondrial Science and Medicine, Michigan State University, East Lansing, MI 48823, USA.
Laurie S. Kaguni, Department of Biochemistry and Molecular Biology and Center for Mitochondrial Science and Medicine, Michigan State University, East Lansing, MI 48823, USA.
Francisco J. Cao, Instituto Madrileño de Estudios Avanzados en Nanociencia, IMDEA Nanociencia. 28049 Madrid, Spain.
Borja Ibarra, Instituto Madrileño de Estudios Avanzados en Nanociencia, IMDEA Nanociencia. 28049 Madrid, Spain.

Document Type

Article

Publication Date

6-20-2019

Subject Area

Bacteriophage T7 (enzymology, genetics); DNA Polymerase gamma (genetics); DNA Replication (drug effects); DNA, Single-Stranded (metabolism); DNA, Viral (metabolism); DNA-Binding Proteins (metabolism); DNA-Directed DNA Polymerase (metabolism); Escherichia coli (genetics); Humans; Kinetics; Nucleic Acid Conformation; Optical Tweezers; Recombinant Proteins; Temperature; Thermodynamics; Viral Proteins (metabolism)

Abstract

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 ∼12 kBT, 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.

Publication Title

Nucleic acids research

Volume

47

Issue

11

First Page

5723

Last Page

5734

Digital Object Identifier (DOI)

10.1093/nar/gkz249

PubMed ID

30968132

E-ISSN

1362-4962

Language

eng

Citation Information

Cerrón, Fernando; de Lorenzo, Sara; Lemishko, Kateryna M.; Ciesielski, Grzegorz L.; Kaguni, Laurie S.; Cao, Francisco J.; and Ibarra, Borja, "Replicative DNA polymerases promote active displacement of SSB proteins during lagging strand synthesis" (2019). UNF Faculty Research and Scholarship. 3285.
https://digitalcommons.unf.edu/unf_faculty_publications/3285