Please cite this article in press as: Riera A, et al. Helicase loading: How to build a MCM2-7 double-hexamer. Semin Cell Dev Biol (2014), http://dx.doi.org/10.1016/j.semcdb.2014.03.008 ARTICLE IN PRESS G Model YSCDB-1526; No. of Pages 6 Seminars in Cell & Developmental Biology xxx (2014) xxx–xxx Contents lists available at ScienceDirect Seminars in Cell & Developmental Biology j ourna l h o me page: www.elsevier.com/locate/semcdb Review Helicase loading: How to build a MCM2-7 double-hexamer Alberto Riera, Silvia Tognetti, Christian Speck * DNA Replication Group, Faculty of Medicine, Institute of Clinical Sciences, Imperial College, Hammersmith Hospital Campus, Du Cane Road, London W12 0NN, UK a r t i c l e i n f o Article history: Available online xxx Keywords: MCM2-7 DNA replication Licensing Pre-RC Helicase a b s t r a c t A central step in eukaryotic initiation of DNA replication is the loading of the helicase at replication origins, misregulation of this reaction leads to DNA damage and genome instability. Here we discuss how the helicase becomes recruited to origins and loaded into a double-hexamer around double-stranded DNA. We specifically describe the individual steps in complex assembly and explain how this process is regulated to maintain genome stability. Structural analysis of the helicase loader and the helicase has provided key insights into the process of double-hexamer formation. A structural comparison of the bacterial and eukaryotic system suggests a mechanism of helicase loading. © 2014 Elsevier Ltd. All rights reserved. Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 2. DNA replicon model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 3. DNA replication origins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 4. Recruitment of the MCM2-7 helicase to the replication origin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 5. The role of ATP-hydrolysis during pre-RC formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 6. Helicase loading – what do we know? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 7. The recruitment of the second MCM2-7 hexamer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 8. ORC/Cdc6 function as a MCM2-7 chaperone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 9. The MCM2-7 double hexamer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 10. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 1. Introduction Precise duplication of the genome is essential for genomic sta- bility and organism survival. As such, cells have evolved highly regulated mechanisms that control DNA replication guarantee- ing the faithful replication of the genome. In all living organisms the replication process is initiated at origins of DNA replica- tion. Eukaryotes employ a six-subunit origin-recognition complex (ORC), shown in Saccharomyces cerevisiae to bind to replica- tion origins [1]. ORC is chromatin bound throughout the cell cycle; however in late M phase Cdc6 binds to ORC to form the ORC/Cdc6 complex [2]. ORC/Cdc6 functions together with Cdt1 to * Corresponding author. Tel.: +44 020 8383 3387. E-mail address: chris.speck@imperial.ac.uk (C. Speck). load the replicative helicase MCM2-7 onto DNA. During helicase loading, also termed pre-replicative complex (pre-RC) formation or DNA licensing, two MCM2-7 hexamers are loaded in an ATP- hydrolysis dependent process into a MCM2-7 double-hexamer around double-stranded DNA [3,4]. Interestingly, this complex is not functional as a helicase and still requires activation in S-phase. Numerous protein factors and kinases, including cyclin-dependent kinase (CDK) and Dbf4 dependent kinase (DDK), act together to promote the formation of a Cdc45/MCM2-7/GINS (CMG) complex, which represents the active form of the replicative helicase [5]. Dur- ing helicase activation the MCM2-7 double-hexamer splits and its ATPase motor becomes activated [6,7]. Within the CMG, MCM2-7 encircles only one strand of DNA, while the other strand is thought to pass through Cdc45 and the four subunit GINS complex, thus enabling the helicase to split the two DNA strands [7,8]. The CMG represents the basis for the replication fork, with DNA polymerases http://dx.doi.org/10.1016/j.semcdb.2014.03.008 1084-9521/© 2014 Elsevier Ltd. All rights reserved.