ZIA BC 010411 (ZIA) | |||
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Title | Initiation of DNA Replication in Mammalian Cells | ||
Institution | NCI, Bethesda, MD | ||
Principal Investigator | Aladjem, Mirit | NCI Program Director | N/A |
Cancer Activity | N/A | Division | CCR |
Funded Amount | $936,196 | Project Dates | 00/00/0000 - 00/00/0000 |
Fiscal Year | 2017 | Project Type | Intramural |
Research Topics w/ Percent Relevance | Cancer Types w/ Percent Relevance | ||
Cancer (100.0%) Gene Therapy (15.0%) |
N/A | ||
Research Type | |||
Cancer Initiation: Oncogenes & Tumor Suppressor Genes Interactions of Genes and/or Genetic Polymorphisms with Exogenous and/or Endogenous Factors |
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Abstract | |||
Within eukaryotic cells, genome duplication initiates at multiple sites on each chromosome. Replication initiation events in diploid mitotic cells proceed in a precise order and are strictly regulated by a series of cell cycle checkpoint signaling pathways. These regulatory constraints, however, are often relaxed in cancer cells. Because the processes that coordinate replication ultimately converge on chromatin, understanding the molecular events that precede DNA replication at the chromatin level is crucial if we are to fully understand cell growth. Critical information about this process is missing because protein complexes that initiate chromosomal replication seem to bind DNA indiscriminately. To gain a complete understanding of the DNA replication process we must resolve how this non-specific DNA binding translates into highly coordinated replication. Our studies are based on the hypothesis that sequence-specific signaling molecules associate with replication initiation sites on chromatin where they modulate the local activity of the ubiquitous replication machinery and dictate both the location and timing of replication initiation events. To test this hypothesis, we characterize protein-DNA interactions at replication initiation sites and identify interactions that play regulatory roles in the DNA replication process. We use two approaches to characterize DNA-protein interactions at replication initiation sites. The first approach utilizes distinct DNA sequences, termed replicators, which facilitate the initiation of DNA replication. We have initially identified these replicator sequences and we now use them as bait to isolate protein complexes that potentially regulate replication. In recent studies we have identified two discrete DNA-protein complexes within one replicator element. One of these complexes includes chromatin remodeling proteins that determine both replication timing and transcriptional activity (Mol Cell Biol. 31:3472-84; 2011). Another complex includes RepID, a member of the DDB1-Cul4-associated-factor (DCAF) family, which binds a subset of replication initiation sites and is required for replication at those sites (Nat Commun. 8;7:11748; 2016). Our studies have demonstrated that RepID associates with chromatin-loop interactions between a replicator element and a distal regulatory sequence within the human beta globin (HBB) locus. We have characterized RepID interactions with other proteins, identified RepID protein partners using a non-biased approach and pinpointed protein domains within RepID that facilitate DNA-protein and protein-protein interactions. Our analyses demonstrate that RepID binding origins require RepID for initiation of DNA replication, providing the first example of a site-specific interaction that determines the initiation of DNA replication on a group of metazoan replication origins. The second approach involves developing tools to map replication initiation sites throughout the genome, and using these tools to analyze DNA replication in the context of chromatin modifications and transcriptional activity. The developed methods involve massively parallel sequencing and single-fiber imaging of replication fork progression. These procedures allow us to study the dynamics of DNA replication at the whole-genome level. Using this methodology we can test whether groups of replication initiation sites share specific properties - for example, if they associate with a particular chromatin feature. We can also identify groups of initiation sites that respond in a similar fashion to a cellular challenge, and test whether distinct groups of replication initiation sites are regulated through association with particular proteins (such as RepID). We have generated a comprehensive dataset of replication initiation sites for several human cancer cell lines (Genome Res. 21:1822-32, 2010; Epigenetics and Chromatin 9:18, 2016). Our recent comprehensive analysis of chromatin modifications associated wit |