The Blow Lab:

Origin Licensing and its Regulation

     Blow J.J. and Laskey, R.A. (1988). A role for the nuclear envelope in controlling DNA replication within the cell cycle. Nature 332, 546-548. [abstract]

     Blow J.J. (1993). Preventing re-replication of DNA in a single cell cycle: evidence for a Replication Licensing Factor. J. Cell Biol. 122, 993-1002. [abstract]

     Chong, J.P.J., Mahbubani, H.M., Khoo, C-Y. and Blow, J.J. (1995). Purification of an Mcm-containing complex as a component of the DNA Replication Licensing system. Nature 375, 418-421. [abstract]

     Rowles, A., Chong, J.P.J., Brown, L., Howell, M., Evan, G.I. and Blow, J.J. (1996). Interaction Between the Origin Recognition Complex and the Replication Licensing System in Xenopus. Cell 87, 287-296. [abstract]

     Tada, S., Li, A., Maiorano, D., Méchali, M. and Blow, J.J. (2001). Repression of origin assembly in metaphase depends on inhibition of RLF-B/Cdt1 by geminin. Nature Cell Biol. 3, 107-113. [abstract]

     Gillespie, P.J., Li, A. and Blow, J.J. (2001). Reconstitution of licensed replication origins on Xenopus sperm nuclei using purified proteins. BioMed Central Biochem. 2, 15. [abstract]

     Li, A. and Blow, J.J. (2005). Cdt1 down-regulation by proteolysis and geminin inhibition prevents DNA re-replication in Xenopus. EMBO J. 24, 395-404. [abstract]

     Blow, J.J. and Dutta, A. (2005). Preventing re-replication of chromosomal DNA. Nat. Rev. Cell Mol. Biol. 6, 476-486. [abstract]

     De Marco V., Gillespie P.J., Li A., Karantzelis N., Christodoulou E., Klompmaker R., van Gerwen S., Fish A., Petoukhov M.V., Iliou M.S., Lygerou Z., Medema R.H., Blow J.J., Svergun D.I., Taraviras S. and Perrakis A. (2009). Quaternary structure of the human Cdt1-Geminin complex regulates DNA replication licensing. Proc. Natl. Acad. Sci. USA 106, 19807-19812.  [abstract]

     Gambus, A., Khoudoli, G.A., Jones, R.C. and Blow, J.J. (2011). Mcm2-7 form double hexamers at licensed origins in Xenopus egg extract. J. Biol. Chem. 286, 11855-64. [abstract]

     Sasaki, T., Li, A., Gillespie, P.J., Blow, J.J. and Gilbert, D.M. (2011). A late-G1 phase inhibitor of replication licensing in mammalian cells that is distinct from geminin or Cdk activity. Nucleus 2, 455-464. [abstract]

     Kisielewska, J. and Blow, J.J. (2012). Dynamic interactions of high Cdt1 and geminin levels regulate S phase in early Xenopus embryos. Development 139, 63-74 [abstract]

     Sonneville, R., Querenet, M., Craig, A., Gartner, A. and Blow, J.J. (2012). The dynamics of replication licensing in live C. elegans embryos. J. Cell Biol. 196, 233-246. [abstract] [pdf]

Neelsen, K.J., Zanini, I.M.Y., Mijic, S., Herrador, R., Chaudhuri, A.R., Creavin, K.D., Blow, J.J. and Lopes M. (2013). Deregulated origin licensing leads to chromosomal breaks by re-replication of a gapped DNA template. Genes Dev. 27, 2537-2542. [abstract] [pdf]

Sonneville, R., Craig, G., Labib, K., Gartner, A. and  Blow, J.J. (2015). Both chromosome decondensation and condensation are dependent on DNA replication in C. elegans embryos. Cell Rep 12, 405-417.  [abstract] [pdf]

Galanos, P., Vougas, K., Walter, D., Polyzos, A., Maya-Mendoza, A., Haagensen, E.J., Kokkalis, A., Roumelioti, F-M., Gagos, S., Tzetis, M., Canovas, B., Igea, A., Ahuja,  A.K., Zellweger, R., Havaki, S., Kanavakis, E., Kletsas, D., Roninson, I., Garbis, S.D., Lopes, M., Nebreda, A., Thanos, D., Blow, J.J., Townsend, P., Sørensen, C.S., Bartek, J., Gorgoulis, V.G. (2016). Chronic p53-independent p21 expression deregulates replication licensing, leading to genomic instability. Nat Cell Biol, 18:777-89. [abstract]

Although we have reconstituted the licensing reaction with purified proteins, we still have only a poor understanding of what it really represents. It now seems likely that during licensing, two ring-shaped Mcm2-7 hexamers are opened up by a clamp loader complex consisting of ORC, Cdc6 and Cdt1, and the Mcm2-7 rings are then closed again around double-stranded DNA. The loading of two hexamers onto DNA provides a configuration potentially capable of initiating a pair of bidirectional replication forks in S phase.  We are currently exploring various aspects of this model. It is known that the multiple copies of Mcm2-7 that are loaded onto each origin end up at sites on DNA distant from ORC and we are testing various models for how this occurs. We and others have described a central role for geminin in preventing re-replication of DNA in metazoans, and we have also shown that the activity of Xenopus geminin is down-regulated by ubiquitination. We are currently examining various aspects of geminin structure to determine how it is affected by ubiquitination and how it is reactivated following nuclear import.