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Giacomo De Piccoli

Summary

The accurate duplication of the genome is essential for the survival of all living cells, and acts as a critical barrier to the development of cancer in humans. Failure to complete DNA replication leads to chromosome breaks, translocation, loss of heterozygosity or cell death. Our lab aims to understand one of the most important pathways through which cells can survive obstacles in DNA replication, known as the S-phase checkpoint.

representation of the replisome at replication fork (A) and activation of the S-phase checkpoint kinases Mec1 and Rad53 following replication stress.Chromosome duplication starts with the assembly on the DNA of the replication machinery, referred to as the replisome. This is composed of a heteromeric DNA helicase called Cdc45-MCM-GINS, three different DNA polymerases, and many regulatory factors (Fig. 1A).

Damage to the DNA template or shortages of dNTPs during DNA replication cause the accumulation of single-stranded DNA at replication forks, which triggers a highly conserved signalling cascade known as the S-phase checkpoint. In budding yeast cells, this involves activation of the Mec1 protein kinase, which in turn initiates the effector kinase Rad53 (functional orthologues of ATR and Chk1, respectively).

The S-phase checkpoint has been shown to play many roles in response to DNA replication stress, including the regulation of cell cycle progression, gene expression, firing of origins of replication, DNA recombination and repair. However, the mechanisms underlying the S-phase checkpoint response at forks are still poorly understood.

Previous work (De Piccoli et al, 2012) has shown that replisome stability is independent of checkpoint kinases, and the focus of our lab is to understand how the checkpoint pathway regulates aspects of replisome function. We have identified novel post-translational modifications of the replisome that are controlled by the S-phase checkpoint pathway, and we will determine whether these control the recruitment of regulatory factors to the replisome, or else directly modulate the activity of key replisome components.

In addition, several components of the replisome itself are required for full activation of the S-phase checkpoint (such as, for example, Tof1, Mrc1, Mcm6 or Pol2), but the molecular details of these roles are still unclear. We are analysing how these replisome components promote activation of the checkpoint response as well as the maintenance of genome stability.

Since the replisome and the S-phase checkpoint response are highly conserved throughout evolution, our ultimate aim is to uncover novel mechanisms of regulation at replication forks that promote genome stability in all eukaryotic cells.

Selected publications:

García-Rodríguez LJ, De Piccoli G, Marchesi V, Jones RC, Edmondson RD, Labib

K. A conserved Polϵ binding module in Ctf18-RFC is required for S-phase

checkpoint activation downstream of Mec1. Nucleic Acids Res. 2015 Oct

15;43(18):8830-8. doi: 10.1093/nar/gkv799.

Maric M, Maculins T, De Piccoli G, Labib K. Cdc48 and a ubiquitin ligase drive

disassembly of the CMG helicase at the end of DNA replication. Science. 2014 Oct

24;346(6208):1253596. doi: 10.1126/science.1253596.

Sengupta S, van Deursen F, de Piccoli G, Labib K. Dpb2 integrates the

leading-strand DNA polymerase into the eukaryotic replisome. Curr Biol. 2013 Apr

8;23(7):543-52. doi: 10.1016/j.cub.2013.02.011.

De Piccoli G, Katou Y, Itoh T, Nakato R, Shirahige K, Labib K. Replisome

stability at defective DNA replication forks is independent of S phase checkpoint

kinases. Mol Cell. 2012 Mar 9;45(5):696-704. doi: 10.1016/j.molcel.2012.01.007.