Initiation of DNA Replication in Mammalian Cells

Division Of Basic Sciences - Nci

Investigators

Linked publications, trials & patents

Abstract

Regulatory constraints that prevent untimely and excessive DNA synthesis and enforce a precise order of chromosome duplication are often relaxed in cancer cells. Understanding the molecular events that precede the onset of DNA synthesis at the chromatin level is crucial if we are to fully understand cell proliferation in health and disease. We study signaling pathways that associate with chromatin to both the location, timing and progression of DNA synthesis. To identify such signaling pathways, we characterize protein-DNA interactions at replication initiation sites (replication origins) and identify interactions that play regulatory roles in the DNA replication process. We are specifically interested in protein-DNA interactions that modify chromatin and affect the cellular response to DNA damage and cancer chemotherapy. In recent studies we have identified DNA-protein complexes that selectively form on distinct groups of replication origins. 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 have shown 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 replication 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 also demonstrated that RepID and the CRL4 complex play a separate, essential role in mitotic cell division. RepID associated complexes are involved in degrading specific proteins that regulate replication, cells without RepID can re-replicate parts of their genome without undergoing mitosis. We have dissected the dynamics of re-replication and identified replication origins that normally interact with the signaling pathways that constrain re-replication, providing insights into an important process that can be triggered by oncogenes and targeted by chemotherapeutic drugs. We have also demonstrated that replication origins bind 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). Dormant replication origins are activated in cells that do active SIRT1, resulting in an increased density of replication initiation events, suggesting that suppression of dormant origins requires SIRT1's deacetylase activity. Cells with activated dormant origins harbor extrachromosomal elements and DNA breaks, suggesting that maintenance of origin dormancy by SIRT1 facilitates genomic stability. We are currently investigating how SIRT1 modulates replication origin activation in cells exposed to stressful conditions. 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 are currently involved in several collaborative studies characterizing replication dynamics following exposure to anti-cancer chemotherapy and chromatin modulators. 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. Our studies are facilitated by tools we have developed to map replication initiation sites throughout the genome and analysis tools that compare replication initiation sites with distinct chromatin features. To facilitate these studies, we are collaborating with others at the CCR to develop tools characterizing genetic and epigenetic features of cancer cells as well as DNA-protein interaction loci. We also participate in collaborative efforts that correlate genetic and epigenetic signatures of specific cancers with their response to therapy.