To successfully pass on its genetic information, every cell must make a perfect duplicate of the genome in every cell cycle. Failure to copy every chromosome faithfully leads to genomic instability, which is the cause of cancer. As a result, replication initiation is strictly regulated, both within the normal cell cycle and after DNA damage.We are interested in how this regulation of DNA replication is achieved in eukaryotes during the cell cycle and when replication forks stall.

Unlike prokaryotes, eukaryotes replicate their genomes from multiple origins.This has the advantage of facilitating the evolution of much larger and more complex genomes, but it does create a problem: If there are multiple origins in the genome, how is origin firing coordinated to make sure that no origin fires more than once?

The assembly of the eukaryotic replication apparatus at origins is tightly regulated in two critical steps.The first step, pre-replicative complex (pre-RC) formation, involves the loading of the replicative helicase Mcm2-7 in an inactive form at origins.This complex can only form in G1 phase of the cell cycle when the APC/C is active and CDK activity is low.This is because CDKs and other APC/C targets such as Geminin are potent inhibitors of pre-RC formation. Once cells enter S-phase, the APC/C is inactivated, CDK activity (and also Geminin) rises and any further pre-RC formation is blocked.

In addition to its role as an inhibitor of pre-RC formation, CDK, together with a second kinase - DDK (Cdc7/Dbf4), are essential for the second step in replication initiation, which involves the activation of the Mcm2-7 helicase and the recruitment of DNA polymerases to origins.We have previously shown that CDK phosphorylates the two essential initiation factors Sld2 and Sld3, which in turn allows binding to another essential initiation factor called Dpb11. How CDK phosphorylation of these targets facilitates replication initiation is not known, but the transient association of these factors at origins has been termed the pre-initiation complex (pre-IC). Since CDK activity both inhibits pre-RC formation and is essential to initiate replication, this produces a switch that only allows replication initiation in S-phase.

Our research is focused on the pre-initiation complex step in the replication reaction.This step is the key CDK regulatory step, but the function of this intermediate is not known. Furthermore, the pre-IC also integrates information from other kinases, such as the DNA damage checkpoint and may be responsible for regulating how efficiently and when an origin fires during S-phase. Much of our understanding of the pre-IC in eukaryotes comes from studies in budding yeast, but how replication initiation is regulated in other eukaryotes is largely unknown. Our aim is to take advantage of the expertise in the wide variety of organisms within the institute and extend these budding yeast studies to the nematode C.elegans and to mammalian cells.

Eukaryotic replication initiation begins with the formation of a pre-replicative complex (pre-RC) on origin DNA during G1 phase. On entry into S-phase, two kinases are activated - CDK and Cdc7/Dbf4. CDK activity contributes to the formation of the pre-initiation complex (pre-IC) which facilitates replication initiation by an unknown mechanism.

Plain English:
For cells to multiply, the DNA template (genome) must be perfectly copied exactly once. This copying process – termed DNA replication – must not only be accurate but also must be completed in its entirety before a cell can divide. Ensuring that the genome is successfully duplicated requires that DNA replication is tightly controlled at multiple levels. In our lab we are interested in understanding these different levels of regulation, since loss of this control leads to defects in genome integrity, which contributes to the onset of cancer in humans.

Selected publications:

• Davide Mantiero, Amanda Mackenzie, Anne Donaldson and Philip Zegerman. Limiting replication initiation factors execute the temporal programme of origin firing in budding yeast. EMBO journal doi:10.1038/emboj.2011.404

• Pagliuca FW, Collins MO, Lichawska A, Zegerman P, Choudhary JS, Pines J. Quantitative proteomics reveals the basis for the biochemical specificity of the cell-cycle machinery. Mol Cell. 2011. 43:406-17.

•Zegerman P and Diffley JF (2010) Checkpoint dependent inhibition of DNA replication initiation via phosphorylation of Sld3 and Dbf4. Nature [Under revision]

• Zegerman P and Diffley JF (2009) DNA replication as a target of the DNA damage checkpoint. DNA repair 8,1077-88

• Zegerman P and Diffley JF (2007) Phosphorylation of Sld2 and Sld3 by cyclin-dependent kinases promotes DNA replication in budding yeast. Nature 445, 281-5

• Zegerman P and Diffley JF (2003) Lessons in how to hold a fork. Nature Struct Biol 10, 778-9

Phospho-proteomic analysis of the Rad53 kinase; Arrays of peptides, representing all the known proteins in the replisome are phosphorylated by the Rad53 kinase in vitro

Replication initiation must be strictly controlled to occur once, and only once, in every cell cycle.

Fig 2: Interactions between Dpb11 and phospho-Sld2/Sld3 in vitro (left panels) are confirmed to be essential for replication initiation in vivo (right panel).