TY - JOUR
T1 - The structural basis of Cdc7-Dbf4 kinase dependent targeting and phosphorylation of the MCM2-7 double hexamer
AU - Saleh, Almutasem
AU - Noguchi, Yasunori
AU - Aramayo, Ricardo
AU - Ivanova, Marina E.
AU - Stevens, Kathryn M.
AU - Montoya, Alex
AU - Sunidhi, S.
AU - Carranza, Nicolas Lopez
AU - Skwark, Marcin J.
AU - Speck, Christian
N1 - Funding Information:
We thank members of the Speck lab, Michael Weinreich, Robert Sclafani and Corrado Santocanale for critical reading of the manuscript. We thank Indiana Magdalou and Audrey Mossler for their help with the expression and purification of proteins. C.S. was funded by the Biotechnology and Biological Sciences Research Council (BB/T005378/1), the Medical Research Council (A652-5PY40) and the Wellcome Trust (107903/Z/15/Z). Y.N. was supported by a JSPS Overseas Research Fellowships from the Japan Society for the Promotion of Science. We thank the LMS and ICL cryo-EM facilities for their support. We thank Holger Kramer and Pavel Shliaha of the LMS Biological Mass Spectrometry and Proteomics Facility for mass-spectrometry and phosphoproteomic analysis. We thank the LonCEM cryo-EM facility funded by Wellcome Trust (206175/Z/17/Z) and Diamond Light Source for access and support of the cryo-EM facilities at the UK national electron bio-imaging centre (eBIC), proposals: EM18659-32 (MD-ATPγS), BI25127-10 (MD-ADP:BeF3) and EM18659-15 (MD-ATP), funded by the Wellcome Trust, MRC, and BBSRC.
Funding Information:
We thank members of the Speck lab, Michael Weinreich, Robert Sclafani and Corrado Santocanale for critical reading of the manuscript. We thank Indiana Magdalou and Audrey Mossler for their help with the expression and purification of proteins. C.S. was funded by the Biotechnology and Biological Sciences Research Council (BB/T005378/1), the Medical Research Council (A652-5PY40) and the Wellcome Trust (107903/Z/15/Z). Y.N. was supported by a JSPS Overseas Research Fellowships from the Japan Society for the Promotion of Science. We thank the LMS and ICL cryo-EM facilities for their support. We thank Holger Kramer and Pavel Shliaha of the LMS Biological Mass Spectrometry and Proteomics Facility for mass-spectrometry and phosphoproteomic analysis. We thank the LonCEM cryo-EM facility funded by Wellcome Trust (206175/Z/17/Z) and Diamond Light Source for access and support of the cryo-EM facilities at the UK national electron bio-imaging centre (eBIC), proposals: EM18659-32 (MD-ATPγS), BI25127-10 (MD-ADP:BeF) and EM18659-15 (MD-ATP), funded by the Wellcome Trust, MRC, and BBSRC. 3
Publisher Copyright:
© 2022, The Author(s).
PY - 2022/12
Y1 - 2022/12
N2 - The controlled assembly of replication forks is critical for genome stability. The Dbf4-dependent Cdc7 kinase (DDK) initiates replisome assembly by phosphorylating the MCM2-7 replicative helicase at the N-terminal tails of Mcm2, Mcm4 and Mcm6. At present, it remains poorly understood how DDK docks onto the helicase and how the kinase targets distal Mcm subunits for phosphorylation. Using cryo-electron microscopy and biochemical analysis we discovered that an interaction between the HBRCT domain of Dbf4 with Mcm2 serves as an anchoring point, which supports binding of DDK across the MCM2-7 double-hexamer interface and phosphorylation of Mcm4 on the opposite hexamer. Moreover, a rotation of DDK along its anchoring point allows phosphorylation of Mcm2 and Mcm6. In summary, our work provides fundamental insights into DDK structure, control and selective activation of the MCM2-7 helicase during DNA replication. Importantly, these insights can be exploited for development of novel DDK inhibitors.
AB - The controlled assembly of replication forks is critical for genome stability. The Dbf4-dependent Cdc7 kinase (DDK) initiates replisome assembly by phosphorylating the MCM2-7 replicative helicase at the N-terminal tails of Mcm2, Mcm4 and Mcm6. At present, it remains poorly understood how DDK docks onto the helicase and how the kinase targets distal Mcm subunits for phosphorylation. Using cryo-electron microscopy and biochemical analysis we discovered that an interaction between the HBRCT domain of Dbf4 with Mcm2 serves as an anchoring point, which supports binding of DDK across the MCM2-7 double-hexamer interface and phosphorylation of Mcm4 on the opposite hexamer. Moreover, a rotation of DDK along its anchoring point allows phosphorylation of Mcm2 and Mcm6. In summary, our work provides fundamental insights into DDK structure, control and selective activation of the MCM2-7 helicase during DNA replication. Importantly, these insights can be exploited for development of novel DDK inhibitors.
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U2 - 10.1038/s41467-022-30576-1
DO - 10.1038/s41467-022-30576-1
M3 - Article
C2 - 35614055
AN - SCOPUS:85130684798
SN - 2041-1723
VL - 13
JO - Nature communications
JF - Nature communications
IS - 1
M1 - 2915
ER -