Johannes Walter

Department of Biological Chemistry and Molecular Pharmacology
Harvard Medical School
Building C2- Room 226A
240 Longwood Avenue
Boston, MA 02115
Tel: (617) 432-4799
Fax: (617) 738-0516
Email: johannes_walter@hms.harvard.edu
Web Page: The Walter Lab Page
6 postdoctoral fellows, 1 student

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 approach 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. 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 that Cdt1 ubiquitylation and destruction are triggered when it associates with the elongating replisome, leading to recruitment of a Cdt1-specific E3 ubiquitin ligase [1]. Being dependent on the assembly of a multi-protein complex (the replisome), this mechanism represents a new paradigm for temporally and spatially-restricted ubiquitylation. We are currently exploring the molecular details of this pathway [4] and whether a similar logic is used to regulate destruction of other substrates.

2. What is the mechanism of the replicative DNA helicase?

One of the most important but poorly understood replication factors is the helicase that unwinds DNA at the replication fork. Our data indicate that in eukaryotes, this enzyme is composed of MCM2-7 (a hexameric ATPase), Cdc45, and GINS [2]. We are using replication of designer DNA templates in egg extracts to understand the mechanism of how this helicase can processively unwind up to 200 kb of DNA.

3. 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 for ICL repair in vertebrates [3] and to determine which step in repair is promoted by two of the Fanc proteins (submitted). We are continuing to use this approach to explore how the 13 Fanc proteins promote DNA repair.

References:

Arias, E.E. and Walter, J.C. (2006). PCNA functions as a molecular platform to trigger Cdt1 destruction and prevent re-replication. Nature Cell Biology 8, 84-90.
Pacek, M., Tutter, A.V., Kubota, Y., Takisawa, H., and Walter, J.C. (2006). Localization of MCM2-7, Cdc45, and GINS to the site of DNA unwinding during eukaryotic DNA replication. Molecular Cell 21, 581-587.
Räschle, R., Knipscheer, P., Enoiu, M., Angelov, T., Sun, J.,, Griffith, J.D., Ellenberger, T.E., Schärer, O.D., and Walter, J.C. (2008). Mechanism of replication-coupled DNA interstrand cross-link repair. Cell 134, 969-980.
Havens, C. and Walter, J.C. (2009). Docking of a specialized PIP box onto chromatin-bound PCNA creates a degron for the ubiquitin ligase CRL4Cdt2. Molecular Cell, in press.

BBS webpage updated 12/02/2009