项目来源

美国卫生和人类服务部基金(HHS)

项目主持人

未公开

项目受资助机构

DIVISION OF BASIC SCIENCES - NCI

项目编号

1ZIABC010411-19

立项年度

2018

立项时间

未公开

项目级别

国家级

研究期限

未知 / 未知

受资助金额

1545289.00美元

学科

Cancer;Genetics;Human Genome

学科代码

未公开

基金类别

INTRAMURAL RESEARCH

关键词

未公开

参与者

ALADJEM, MIRIT

参与机构

NATIONAL CANCER INSTITUTE

项目标书摘要：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 started by identifying 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 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. We found that RepID associates with chromatin-loop interactions between a replicator element and a distal regulatory sequence within the human beta globin (HBB) locus. Our recent studies show that RepID exerts its effects on replication by recruiting a ubiquitin ligase complex, CRL4, to chromatin, suggesting that ubiquitin ligase complexes play a role in regulating DNA replication dynamics. Importantly, 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. We have shown that replication origins bind another protein, a phosphorylated form of the NAD+-dependent deacetylase SIRT1. Unlike RepID, SIRT1 is not required for initiation of DNA replication, and instead, it prevents replication from initiating in a subgroup of potential origins (dormant origins). In concordance, dormant replication origins are activated, and the overall frequency of replication initiation events increases, in cells that do not contain the phosphorylated form of SIRT1 (either due to a depletion or to a mutation in the phosphorylation site). We have also observed an increased frequency of replication initiation events in cells that contain a SIRT1 mutant in which deacetylase activity is inactivated, suggesting that suppression of dormant origins requires SIRT1's deacetylase activity. Our studies demonstrate that cells with activated dormant origins harbor extrachromosomal elements and DNA breaks, suggesting that maintenance of origin dormancy by SIRT1 facilitates genomic stability. Our studies are facilitated by tools we have developed to map replication initiation sites throughout the genome, and using these maps to analyze DNA replication in the context of chromatin modifications and transcriptional activity. Using a combination of DNA sequencing and single fiber analyses, have generated a comprehensive dataset of replication initiation sites for several human cancer cell lines. We have demonstrated that replication origin usage varies with tissue type, with distinct modifications associated with cell-type specific replication origins. To facilitate these studies, we have developed a web-based tool (Coloweb) to help decipher the relationships among RepID binding sites and epigenetic features. This tool is available to the community to support bioinformatics characterization of DNA-protein interaction loci. An important aspect of our work pertinent to human health is the response of the replication machinery to perturbations. A large and increasing number of anti-cancer drugs target DNA replication or interfere with cell cycle signaling. Understanding specific cell cycle defects in different cancers is likely to provide clues regarding their sensitivity to anti-cancer therapies. We are currently studying replication origins activated in response to those drugs, directly mapping chromatin targets involved in preventing excess replication. Our strategy consists of combining genome-scale sequencing with single-fiber analyses. This approach can provide important insights into the organization of replication initiation events and the cellular responses to signals that might perturb DNA replication. We ask how particular replication and repair pathways affect the pace and frequency of DNA replication. We observed that a DNA repair endonuclease, Mus81, modulates the pace of DNA replication in the absence of exogenous stress and that its presence is essential to help cells restore DNA synthesis in the presence of drugs that slow replication. We are currently involved in several collaborative studies characterizing replication dynamics following exposure to anti-cancer chemotherapy. In the future, we will investigate how protein-DNA interactions that are required for DNA replication are modulated in response to environmental challenges and anti-cancer drugs. As we learn more about local and distal interactions that promote DNA replication, we will continue to explore pathways that signal back from chromatin to the cell cycle machinery to affect the replication landscape and modulate the response to anti-cancer therapy.