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Research Summary

Maintenance of genome stability: DNA replication and response to DNA damage

Much of our work focuses on the mechanisms that control the proper order, timing and accuracy of cell cycle phases. These mechanisms are essential to maintain the integrity of the genome and the fidelity of transmission of the genetic material throughout cell generations. We use the eggs and embryos of the frog Xenopus laevis and cell-free extracts derived from these as a simple model system to gain important molecular and biochemical insights on cell cycle and checkpoints regulation. In addition, we use mammalian cells in culture as well as mouse models to study some of the biological responses to DNA damage.

Using these model systems we are addressing the following questions:

A) How is the ordered progression of the cell cycle phases maintained, how is DNA replication restricted to once per cell cycle? We are conducting biochemical studies to understand how the proteins at the origins of replication (pre-replicative complex or pre-RC) are assembled and how they function in a cell cycle regulated manner.

B) How is the cell cycle regulated following DNA damage? We are studying the function of the ATM (Ataxia Telangiectasia Mutated) protein. In particular, we have established a novel cell-free system that allows us to study the biochemical role of ATM in vitro.

C) What are the mechanisms that regulate exit from mitosis and chromosomes segregation? We are investigating the molecular nature of the checkpoints that prevent inappropriate chromosome segregation. We are also investigating the cross talk between protein kinase A (PKA) and the cyclin-dependent kinases that regulate mitosis.

D) We are studying the biochemical reactions involved in the repair of several types of DNA damage using Xenopus cell-free extracts.

Dupre A, Boyer-Chatenet L, Gautier J (2006). Two-step activation of ATM by DNA and the Mre11-Rad50-Nbs1 complex. Nat Struct Mol Biol, 13(5): 451-457.

Shechter D, Gautier J (2005). ATM and ATR checks in on origins: a dynamic model for origin selection and activation. Cell Cycle, 4(2): 235-238.

Ying CY, Gautier J (2005). The ATPase activity of MCM2-7 is dispensable for pre-RC assembly but is required for DNA unwinding. Embo J 24(24): 4334-4344.

DiVirgilio M, Gautier J (2005). Repair of double-strand breaks by nonhomologous end joining in the absence of Mre11. J Cell Biology, 171(5): 765-771.

Costanzo V., Paull T., Gottesman M. and Gautier J. (2004). Mre11 assembles linear DNA fragments into DNA damage signaling complexes. PLoS Biology. 2, 600-609 (full text).

Shechter D. Costanzo V. and Gautier J. (2004). Regulation of DNA replication by ATR: signaling in response to DNA intermediates. DNA Repair (Amst), 3(8-9), 901-908.

Costanzo, V., Shechter, D., Lupardus, P.J., Cimprich, K.A., Gottesman, M. and Gautier, J. (2003) An ATR- and Cdc7-dependent checkpoint that inhibits initiation of DNA replication. Molecular Cell 11, 203-213 (full text).

1. DNA damage response and DNA replication
Our long-term objective is to understand the mechanisms by which the different facets of the DNA damage response are integrated within cell cycle progression at the time of DNA replication. The ability to undergo DNA replication in the presence of DNA damage, called Radio-Resistant DNA Synthesis (RDS), is a hallmark of the cellular phenotypes of cancer-prone disorder as well as of tumor cells. We have established a cell-free system derived from Xenopus eggs that recapitulates different aspects of the DNA damage response. In particular, we have been able to identify a novel ATM- dependent cell cycle checkpoint that prevents initiation of DNA replication. We will determine whether the Xenopus homologues of Chk1 and/or Chk2/Cds1 are components of this pathway. We will also determine whether Wee1, Myt1 and/or Cdc25 are components of the pathway. We will take advantage of this cell-free system to identify which of damages can elicit a checkpoint in vitro and whether such responses are ATM or ATR-dependent. Finally, we will examine how ATM and Mre11 complex participate in the coordinated and harmonious response to DNA damage and how cell cycle arrest is integrated DNA repair.
National Cancer Institute
4/2002-3/2007

2. Probing the DNA damage response in vitro
Our long-term objective is to understand the molecular nature of the signal transduction
pathways activated following DNA damage. To that goal, we have established unique cell-free systems derived from Xenopus eggs that faithfully recapitulate several aspects of the DNA damage response. We will use these cell-free systems to screen for proteins that are modified in presence of double strand breaks (DSBs). Next, we will investigate the role in the DNA response of a candidate gene we have identified in a pilot screen and of additional genes we will isolate. We will screen a chemical library for compounds that interfere with the in vitro activation of ATM/ATR protein kinases, an early step of the DNA damage response. The compounds identified will be further validated and their activity will be tested in a variety of assays performed in Xenopus cell-free systems.
National Cancer Institute
9/2002-8/2007

1. Cell cycle regulation of DNA replication
The main focus of this proposal is on the function(s) of the MCM proteins. The 6 MCM proteins form large complexes that are essential for DNA replication. MCM protein complexes are the targets of multiple phosphorylations by cell cycle regulated protein kinases. Our primary objective is to understand the exact function of MCM protein complex(s) during DNA replication and how cell-cycle-dependent regulation influences the activity and the role of these proteins. We are developing cell-free systems and in vitro systems to further study the biochemical function of the MCM proteins and their regulation during the cell cycle.
American Cancer Society
1/2002-12/2005

2. Cell cycle control of the cAMP signal transduction pathways
We will analyze how PKA controls DNA replication in particular the regulation and the activity of cdc6 protein and of the mini chromosome maintenance proteins, required for initiation of DNA replciation. We will determine how cdc2 protein kinase increases cAMP levels and why this is blocked at the mitosis exit checkpoint. Finally, we will study PKA induction of cyclin degradation using defined components of both the cAMP-PKA and the anaphase promoting complex pathways.
National Institute of General Medical Sciences
5/1999-4/2004