A bipartite GINS binding mode of TopBP1 activates the replicative helicase MCM
Matthew Day*2, Bilal Tetik*1, Milena Parlak*1, Yasser Almeida-Hernández3, Elsa Sanchez-Garcia3, Farnusch Kaschani4, Markus Kaiser4, Markus Räschle5, Heike Siegert1, Anika Marko1, Isabel A. Barker2, Laurence H. Pearl2+, Antony W. Oliver2+, Dominik Boos1+
1 Molecular Genetics II, Vertebrate DNA Replication, Centre of Medical Biotechnology, University of Duisburg-Essen, Universitätsstraße 2-5, 45141 Essen, Germany
2 Cancer Research UK DNA Repair Enzymes Group, Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Falmer, Brighton BN1 9RQ, UK
3 Computational Biochemistry, Centre of Medical Biotechnology, University of Duisburg-Essen, Universitätsstraße 2-5, 45141 Essen, Germany
4 Analytics Core Facility Essen, Centre of Medical Biotechnology, University of Duisburg-Essen, Universitätsstraße 2-5, 45141 Essen, Germany
5 Molecular Genetics, Technical University Kaiserslautern, Paul-Ehrlich Straße 24, 67663 Kaiserslautern, Germany
* equal contribution
+ corresponding author
In dividing cells genome duplication is tightly coordinated with the cell cycle to ensure that each daughter cell receives a full copy of the genome. In all eukaryotes DNA replication initiates at replication origins in a two-step process; replication licensing and firing. During G1, licensing factors load the replicative MCM helicase onto DNA. In these pre-Replication complexes (pre-RCs), MCM binds DNA as an inactive symmetrical double hexamer encircling both strands of the double helix. As cells enter S phase, the rising activities of the CDK and DDK cell cycle kinases trigger multiple reconfigurations at origins to activate the MCM helicase and to form two mature bidirectional replisomes, one for each MCM hexamer in the pre-RCs.
While replication licensing is understood well at the molecular level, relatively little is known about the steps that turn MCM into an active replisome. In this process, the “firing factor” TopBP1 plays an essential role by facilitating recruitment of the GINS complex to the MCM helicase, which is key to its activation. To gain insight into TopBP1 function, we solved a cryo-EM structure of a TopBP1 fragment bound to the GINS complex. The structure reveals two separate GINS binding sites within TopBP1, which we validate by mutational analysis, fluorescence anisotropy measurements and MS crosslinking experiments. Using an in vitro replication system based on Xenopus egg extracts, we show that both GINS binding sites in TopBP1 contribute to DNA replication and must cooperate to support full assembly of replisomes from pre-RCs, as shown by comprehensive MS analysis of the chromatin bound replication complexes.
Our studies provide novel molecular insight into replisome. They further offer a basis for understanding how TopBP1 performs it essential function in replication origin firing and how this is coordinated with TopBP1’s plethora of functions in the DNA damage response and chromosome maintenance.
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