Research Interests:

DNA replication and repair mechanisms that suppress genomic instability

Xenopus laevis egg extracts have an extraordinary capacity to recapitulate key events of cell cycle progression in vitro, including DNA replication and repair, mitosis, and checkpoints.  We are using this system to elucidate the molecular mechanisms that underlie the faithful transmission of genetic information from one cell generation to the next.  We are interested in the following areas:

1.  Single molecule studies of eukaryotic DNA replication.  We recently developed a novel imaging approach that allows us to visualize the movement of single DNA replication forks in real time (Yardimci et al., Molecular Cell, 2010;  Kochaniak et al., submitted).  We are now using this approach to tackle numerous problems in DNA replication and repair, including the ones listed below.

2.  A new mechanism for cell cycle regulated proteolysis 
DNA replication is limited to a single round per cell cycle because a key replication initiation factor, Cdt1, is destroyed in S phase by ubiquitin-mediated proteolysis.  Surprisingly, we found Cdt1 destruction is directly coupled to the process of DNA replication (Arias and Walter, Nature Cell Biology, 2006;  Jin et al, Molecular Cell, 2006).  We are currently exploring the molecular mechanism underlying this coupling (Havens and Walter, Molecular Cell, 2009) and we have been identifying new substrates of this pathway (Centore et al., Molecular Cell, 2010).

3.  What is the mechanism of the replicative DNA helicase? 
One of the most important but poorly understood replication factors is the ‘CMG’ complex, the helicase that unwinds DNA at the replication fork.  Before S phase, CMG loads onto double-stranded (ds)DNA in an inactive form.  We showed that when CMG unwinds DNA in S phase, it translocates along ssDNA, implying that CMG activation involves remodeling from a dsDNA to a ssDNA binding mode (Fu et al., Cell, 2011).  In the future, we will seek to understand the mechanism underlying this remodeling.

4.  Understanding the human disease Fanconi anemia.  Fanconi anemia (FA) is a human cancer predisposition syndrome caused by defects in any one of 13 'Fanc' proteins.  FA cells are extremely sensitivity to agents that cause DNA interstrand cross-links (ICLs), suggesting that Fanc proteins participate in ICL repair. Using Xenopus egg extracts, we developed a cell-free system that supports ICL repair in vitro.  We used this approach to describe the first molecular mechanism of ICL repair in vertebrates (Raschle et al. Cell, 2008) and to determine which step in repair is promoted by two of the Fanc proteins (Knipscheer et al., Science, 2009; Long et al. Science, 2011).  We are continuing to use this approach to explore how the 13 Fanc proteins promote DNA repair.

Selected Publications:

Yardimci, H., Kochaniak, A.B., Habuchi, S., van Oijen, A.M., Walter, J.C.  (2010).  Uncoupling of sister replisomes during eukaryotic DNA replication.  Molecular Cell 40, 834-40.

Long, D.T., Räschle, M., Joukov, V., and Walter J.C. (2011).  Mechanism of Rad51-dependent DNA interstrand cross-link repair.  Science 333, 84-7.

Fu,Y.V., Yardimci, H., Long, D.T., Ho, T.V., Guainazzi, A., Bermudez, V.P., Hurwitz, J., van Oijen, A.M., Schärer, O.D., and Walter, J.C.  (2011).  Selective bypass of a lagging strand roadblock by the eukaryotic replicative DNA helicase.  Cell 146, 931-941.