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DNA synthesis determines the binding mode of the human mitochondrial single-stranded DNA-binding protein

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Morin, José A. and Cerron Campoo, Fernando and Jarillo Díaz, Javier and Beltrán de Heredia Rodríguez, Elena and Ciesielski, Grzegorz L. and Arias González, J. Ricardo and Kaguni, Laurie S. and Cao García, Francisco Javier and Ibarra, Borja (2017) DNA synthesis determines the binding mode of the human mitochondrial single-stranded DNA-binding protein. Nucleic acids research, 45 (12). pp. 7237-7248. ISSN 0305-1048

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Official URL: http://dx.doi.org/10.1093/nar/gkx395


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Abstract

Single-stranded DNA-binding proteins (SSBs) play a key role in genome maintenance, binding and organizing single-stranded DNA (ssDNA) intermediates. Multimeric SSBs, such as the human mitochondrial SSB (HmtSSB), presentmultiple sites to interact with ssDNA, which has been shown in vitro to enable them to bind a variable number of single-stranded nucleotides depending on the salt and protein concentration. It has long been suggested that different binding modes might be used selectively for different functions. To study this possibility, we used optical tweezers to determine and compare the structure and energetics of long, individual HmtSSB-DNA complexes assembled on preformed ssDNA and on ssDNA generated gradually during 'in situ' DNA synthesis. We show that HmtSSB binds to preformed ssDNA in two major modes, depending on salt and protein concentration. However, when protein binding was coupled to strand-displacement DNA synthesis, only one of the two binding modes was observed under all experimental conditions. Our results reveal a key role for the gradual generation of ssDNA in modulating the binding mode of a multimeric SSB protein and consequently, in generating the appropriate nucleoprotein structure for DNA synthetic reactions required for genome maintenance.


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© Oxford University Press 2017.
Spanish Ministry of Economy and Competitiveness [MAT2015-71806-R to J.R.A-G, FIS2010-17440, FIS2015-67765-R to F.J.C., BFU2012-31825, BFU2015-63714-R to B.I.]; Spanish Ministry of Education, Culture and Sport [FPU13/02934 to J.J., FPU13/02826 to E.B-H.]; National Institutes of Health [GM45925 to L.S.K.]; University of Tampere (to G.L.C.); Programa de Financiacion Universidad Complutense de Madrid-Santander Universidades [CT45/15-CT46/15 to F.C.]. Funding for open access charge: Spanish Ministry of Economy and Competitiveness [BFU2015-63714-R].

Uncontrolled Keywords:T4 gene 32 protein; Coli SSB protein; Escherichia coli; Functional interactions; Replication restart; Crystal structure; SSDNA complexes; Reca protein; Polymerase; Displacement.
Subjects:Sciences > Physics > Nuclear physics
ID Code:44820
Deposited On:30 Oct 2017 11:22
Last Modified:10 Dec 2018 14:57

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