Establishment of a Replication Timing Program in Mammalian Cells

Suny, Upstate Medical University, Syracuse NY

Investigators

Abstract

The overall objective of this project is to elucidate how the timing program for the replication of chromosomal DNA is established. It is proposed that repositioning of sequences in early G1-phase creates microdomains that establish thresholds for the initiation of replication. Specific hypotheses derived from this model will be tested. The first project will be to examine whether the re-positioning of sequences in the nucleus is associated with a remodeling of chromatin. This work will determine whether the differential DNaseI accessibility of early- and late-replicating chromatin is established in early G1-phase. It will also examine whether hypoacetylation of histones or association with the chromo-domain protein HP1 is necessary to establish or maintain the late-replication time of heterochromatin. A second project will test the hypothesis that chromosome architecture mediates replication timing by setting thresholds that restrict the access of initiation proteins to a subset of origins, focusing on mammalian homologues of Cdc45 and the Cdc7/Dbf4 complex. These replication initiation proteins have been implicated as key targets for regulating initiation events during S-phase progression in S. cerevisiae. This project will examine whether over-expression of these proteins can advance the replication timing program in mammalian cells. The final project will use a proteomics approach, combined with an in vitro replication timing assay, to identify novel candidates for proteins that establish a replication timing program. The working model predicts that intranuclear microenvironments are created through the association of specific proteins with chromatin. This project will identify those proteins that associate with chromatin during the relatively brief period of domain re-positioning. It will also exploit a cell-free system to enrich for those proteins essential to the establishment of a replication timing program. Proteomics offers a potentially powerful means to approach complex biological problems that do not lend themselves easily to traditional genetic and biochemical approaches. Hence, this project will also test the power of this novel technology. How is the structure of chromosomes related to the functions that they have to carry out in the cell? Chromosomes can be divided into independent domains. When they duplicate in a process called DNA replication, there is a defined order in which the domains are replicated. Furthermore, all DNA within a single domain is replicated simultaneously, coordinated by starting replication synchronously from many sites within the domain. Intriguingly, domains that are not in use in any given cell-type are replicated last, while those that are active are replicated first and changes in the order in which these domains are replicated take place at key stages during the development of organisms. The relationship between changes in the replication timing of domains and the functions of those domains during development has been very difficult to study, due to the lack of tools. Recently, a system in which replication of chromosomes can be carried out in a test tube was developed and shown to accurately recapitulate the replication program of chromosome domains. These studies have opened up an exciting new avenue of research into the relationship between gene expression, chromosome structure and DNA replication. This project will test the hypothesis that proteins previously thought to regulate the structure of chromosomal domains may directly regulate the replication program of those domains.