Student Theses and Dissertations

Date of Award

2011

Document Type

Thesis

RU Laboratory

Chait Laboratory

Keywords

eukaryotic replication forks, Saccharomyces cerevisiae, yeast polymerases, fork dynamics

Abstract

In eukaryotic organisms, each chromosome must be precisely replicated every time a cell divides so that the genetic material can be passed on to the cell’s progeny. The work presented here is an in-depth investigation into the dynamics of the proteins that associate with progressing replication forks in yeast. A focused proteomics approach is employed to specifically identify interactions between the replication fork-coupled GINS complex and other components of the replication machinery. The scope of this technique is extended by applying it to cells that have been synchronized within the cell cycle – revealing the cell cycle dependent interactions of the GINS complex. The results show that GINS is a stable complex throughout the cell cycle, and interacts with components of the replicative helicase and chromatin during S –phase. Previous studies have led to a picture wherein the replication of DNA progresses at variable rates over different parts of the budding yeast genome. It is widely held that the dynamics of replication fork progression are strongly affected by local chromatin structure/architecture, and by interaction with machineries controlling transcription, repair and epigenetic maintenance. Here we adopted a complementary approach to those previously applied for assaying replication dynamics wherein we used whole genome time-resolved ChIP-chip analysis of three integral members of the replication fork – the GINS complex, Polymerase !, and Polymerase ". Surprisingly, our data demonstrate that these proteins progress at highly uniform rates regardless of genomic location, revealing that replication fork dynamics in yeast is simpler and more uniform than previously envisaged. In addition, we demonstrate how the synergistic use of experiment and modeling leads to novel biological insights. In particular, a parsimonious model allowed us to accurately simulate fork movement throughout the genome and also revealed a subtle phenomenon, which we interpret as arising from low frequency fork arrest. Taken together, these experiments suggest that the progressing replication forks take precedence in the genome, and that chromatin state does not have as significant affect on the rate of fork progression, as was previously believed.

Comments

A thesis presented to the faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy.

Permanent URL

http://hdl.handle.net/10209/395

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