The eukaryotic DNA replication protein Mcm10 (mini-chromosome maintenance 10) associates with chromatin in early S-phase and is required for assembly and function of the replication fork protein machinery. Another essential component of the eukaryotic replication fork is Cdc45 (cell division cycle 45), which is required for both initiation and elongation of DNA replication. In the present study we characterize, for the first time, the physical and functional interactions of human Mcm10 and Cdc45. First we demonstrated that Mcm10 and Cdc45 interact in cell-free extracts. We then analysed the role of each of the Mcm10 domains: N-terminal, internal and C-terminal (NTD, ID and CTD respectively). We have detected a direct physical interaction between CTD and Cdc45 by both in vitro co-immunoprecipitation and surface plasmon resonance experiments. On the other hand, we have found that the interaction of the Mcm10 ID with Cdc45 takes place only in the presence of DNA. Furthermore, we found that the isolated ID and CTD domains are fully functional, retaining DNA-binding capability with a clear preference for bubble and fork structures, and that they both enhance Cdc45 DNA-binding affinity. The results of the present study demonstrate that human Mcm10 and Cdc45 directly interact and establish a mutual co-operation in DNA binding.
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September 2013
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Research Article|
August 09 2013
The physical interaction of Mcm10 with Cdc45 modulates their DNA-binding properties Available to Purchase
Roberta Di Perna;
Roberta Di Perna
*Istituto di Biochimica delle Proteine, Consiglio Nazionale delle Ricerche, Via P. Castellino 111, 80131 Napoli, Italy
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Valentina Aria;
Valentina Aria
*Istituto di Biochimica delle Proteine, Consiglio Nazionale delle Ricerche, Via P. Castellino 111, 80131 Napoli, Italy
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Mariarosaria De Falco;
Mariarosaria De Falco
*Istituto di Biochimica delle Proteine, Consiglio Nazionale delle Ricerche, Via P. Castellino 111, 80131 Napoli, Italy
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Vincenzo Sannino;
Vincenzo Sannino
*Istituto di Biochimica delle Proteine, Consiglio Nazionale delle Ricerche, Via P. Castellino 111, 80131 Napoli, Italy
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Andrei L. Okorokov;
Andrei L. Okorokov
†Wolfson Institute for Biomedical Research, University College London, Gower Street, London WC1E 6BT, U.K.
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Francesca M. Pisani;
Francesca M. Pisani
1
*Istituto di Biochimica delle Proteine, Consiglio Nazionale delle Ricerche, Via P. Castellino 111, 80131 Napoli, Italy
1Correspondence may be addressed to either of these authors (email [email protected] or [email protected]).
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Mariarita De Felice
Mariarita De Felice
1
*Istituto di Biochimica delle Proteine, Consiglio Nazionale delle Ricerche, Via P. Castellino 111, 80131 Napoli, Italy
1Correspondence may be addressed to either of these authors (email [email protected] or [email protected]).
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Publisher: Portland Press Ltd
Received:
January 10 2013
Revision Received:
June 06 2013
Accepted:
June 10 2013
Accepted Manuscript online:
June 10 2013
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2013 Biochemical Society
2013
Biochem J (2013) 454 (2): 333–343.
Article history
Received:
January 10 2013
Revision Received:
June 06 2013
Accepted:
June 10 2013
Accepted Manuscript online:
June 10 2013
Citation
Roberta Di Perna, Valentina Aria, Mariarosaria De Falco, Vincenzo Sannino, Andrei L. Okorokov, Francesca M. Pisani, Mariarita De Felice; The physical interaction of Mcm10 with Cdc45 modulates their DNA-binding properties. Biochem J 1 September 2013; 454 (2): 333–343. doi: https://doi.org/10.1042/BJ20130059
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