Structure of the replication regulator Sap1 reveals functionally important interfaces.pdf (1.55 MB)
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Structure of the replication regulator Sap1 reveals functionally important interfaces.

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journal contribution
posted on 25.06.2019, 10:11 by MM Jørgensen, B Ekundayo, M Zaratiegui, K Skriver, G Thon, T Schalch
The mechanism by which specific protein-DNA complexes induce programmed replication fork stalling in the eukaryotic genome remains poorly understood. In order to shed light on this process we carried out structural investigations on the essential fission yeast protein Sap1. Sap1 was identified as a protein involved in mating-type switching in Schizosaccharomyces pombe, and has been shown to be involved in programmed replication fork stalling. Interestingly, Sap1 assumes two different DNA binding modes. At the mating-type locus dimers of Sap1 bind the SAS1 sequence in a head-to-head arrangement, while they bind to replication fork blocking sites at rDNA and Tf2 transposons in a head-to-tail mode. In this study, we have solved the crystal structure of the Sap1 DNA binding domain and we observe that Sap1 molecules interact in the crystal using a head-to-tail arrangement that is compatible with DNA binding. We find that Sap1 mutations which alleviate replication-fork blockage at Tf2 transposons in CENP-B mutants map to the head-to-tail interface. Furthermore, several other mutations introduced in this interface are found to be lethal. Our data suggests that essential functions of Sap1 depend on its head-to-tail oligomerization.

Funding

We acknowledge the Swiss Light Source at the Paul Scherrer Institut, Villigen (SLS), Switzerland for provision of synchrotron radiation facilities. We thank Vincent Olieric and Aaron Finke for assistance in using beam line PXIII, Chris Lima for supplying expression vectors, and Eishi Noguchi for the S. pombe sap1-3FLAG-kanR strain. This work was supported by the Swiss National Science Foundation SNF Professorship grants [PP00P3_139137, PP00P3_163760_1, PP00P3_172904 to T.S.]; Fondation Ernst et Lucie Schmidheiny, Fonds Constantin Topali and Société Académique de Genève (T.S.); the Institute for Genetics and Genomics Geneva (IG3) [PhD studentship to B.E.]; the Novo Nordisk Foundation [110233 to G.T. and K.S.]; the Carlsberg Foundation (G.T.); and the National Institute of General Medical Sciences at the National Institute of Health [R01GM105831 to M.Z.].

History

Citation

Scientific Reports, 2018, 8:10930

Author affiliation

/Organisation/COLLEGE OF LIFE SCIENCES/Biological Sciences/Molecular & Cell Biology

Version

VoR (Version of Record)

Published in

Scientific Reports

Publisher

Nature Research (part of Springer Nature)

eissn

2045-2322

Acceptance date

04/07/2018

Copyright date

2018

Available date

25/06/2019

Publisher version

https://www.nature.com/articles/s41598-018-29198-9

Notes

Coordinates and structure factors have been deposited in the Protein Data Bank under accession codes 6EXU (arsenic) and 6EXT (native). Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-29198-9

Language

en

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