The chromosome replication cycle
My lab uses a model cell-free system derived from Xenopus eggs to study how genetic information is faithfully transmitted from one cell generation to the next. We are interested in the following questions:
1. How is DNA replication coordinated with the cell cycle?
DNA replication is restricted to a single round per cell cycle because Cdt1, a key replication initiation protein, is destroyed after the first round of DNA replication. We have discovered that Cdt1 destruction is strictly dependent on the first round of DNA replication, and that it occurs via an unusual mechanism (Ref. 1): Cdt1 and a specific E3 ubiquitin ligase dock onto the replication fork via the replication factor PCNA, leading to ubiquitin transfer to Cdt1, and proteasome-mediated Cdt1 destruction. Our current goal is to understand how PCNA orchestrates Cdt1 ubiquitylation, since this should teach us important lessons about the spatial and temporal regulation of proteolysis.
2. What is the mechanism of the replicative DNA helicase?
Our data indicate that a complex of MCM2-7, Cdc45, and GINS comprises the DNA helicase, which unwinds DNA at the replication fork (Ref. 2). We are using biochemical, ultrastructural (EM), and ChIP approaches to determine how it is possible that this helicase can processively unwind up to 200 kb of DNA.
3. How is sister chromosome cohesion established? Cohesin is a ring-shaped protein complex that holds sister chromatids together, perhaps by forming a molecular “hoola-hoop” that encircles sisters. We recently discovered that the binding of cohesins to chromosomes is intimately linked to the initiation of DNA replication (Ref. 3). We are using this observation to elucidate the mechanism by which cohesion between sisters is established.
4. How are inter-strand DNA cross-links repaired? Inter-strand DNA cross-links are highly toxic DNA lesions because they block passage of RNA and DNA polymerases. We have found that addition of inter-strand DNA cross-links to Xenopus egg extracts activates the Fanconi anemia pathway, which comprises the "first responder" to these DNA lesions in cells. We are taking advantage of this system to understand the molecular mechanism of how Fanconi anemia proteins repair interstrand DNA cross-links.
|
Harvard University Home / GSAS Home / HMS Home 
