How do changes in chromatin structure and cellular differentiation influence the composition and activity of the replication machinery?

Drosophila melanogaster provides an ideal model to understand how the process is DNA replication is modulated during development. Depending on the developmental context, genome duplication can be between three minutes to 12 hours. Furthermore, key changes in chromatin structure and activity are occurring simultaneously, many of which have been characterized at the molecular level. We are exploiting these developmentally programmed changes in the DNA replication program to understand 1) How the replication machinery deals with conflicts with the transcription machinery and R loops. 2) how the machinery responsible for duplication of the genetic and epigenetic material deals with the plethora of changes in chromatin structure throughout the genome and 3) how cell-type-specific changes in the DNA replication program influences the composition and activity of the replication machinery.

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How does Rif1 control replication timing during development?

Rif1 has been shown to control the replication timing program from yeast to humans, but the precise mechanism through which Rif1 acts to control replication timing still remains elusive. Using Drosophila melanogaster as a model system, we have found that Rif1 associates with the replication machinery in a developmentally programmed manner (Munden et al., eLife). We are combining the powerful developmental systems of Drosophila, with biochemical and proteomic approaches to determine how Rif1 associates with the replication machinery. We are also using genomic approaches to determining the effect Rif1 has on the replication timing program in multiple developmental contexts (Armstrong, Das, et al., Genetics). Our goal is to provide mechanistic insight into how Rif1 regulates DNA replication during development.

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How are specific regions of the genome repressed for DNA replication?

During the Drosophila endo cycle specific regions of the genome become repressed for DNA replication, resulting in reduced copy number or underreplication. Repression of DNA replication is mediated, in large part, by the SNF2-domain-containing protein SUUR. We have shown that SUUR represses DNA replication by directly inhibiting the machinery responsible for duplicating the genome. We still don't understand, however, how SUUR is recruited the replication machinery and the molecular mechanism by which it exerts in inhibitory action. We are currently trying to identify how SUUR functions to better understand fundamental properties of the DNA replication program. 

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What influence does chromatin structure have on chromosome fragility?

Within all of our genomes are common fragile sites, which are regions of the genome that are difficult to replicate and prone to breakage upon replication stress. While much work has characterized these sites in different cell types, we still don't understand what the role chromatin structure has on driving chromosome fragility. 

We have mapped and characterized common fragile sites in the Drosophila genome. Next, we plan identify the role chromatin structure plays in promoting chromosome fragility within these common fragile sites.