Initiation of DNA Replication in Mammalian Cells

Division Of Basic Sciences - Nci

Investigators

Linked publications, trials & patents

Abstract

Our studies focus on cellular signaling pathways that regulate the location, timing and progression of DNA synthesis. We have identified cis-acting elements that facilitate the initiation of DNA replication, generated whole-genome-scale maps of replication initiation sites in human cells and detected novel protein-DNA interactions at replication initiation sites (replication origins). These discoveries were enabled by novel bioinformatics and biochemical approaches that we developed and implemented. Mapping replication origin activity and characterizing replication fork progression have demonstrated strong links between replication, histone modifications and chromatin packaging. The observed highly orchestrated order of the DNA replication program, however, contrasts with the low sequence-specificity exhibited by the molecular machines that catalyze DNA synthesis and the absence of a "consensus" DNA sequence that identifies all replication origins. We address this challenge by proposing and testing the hypothesis that genome duplication is guided by nuanced, selective protein-DNA interactions at discrete groups of replication origins that share distinct features. In the recent review period, we used a combination of genetic, biochemical, bioinformatics, imaging and functional analyses to study DNA-protein interactions and chromatin transactions that govern the initiation of DNA replication. We identified protein complexes that selectively assemble on groups of replication origins and modify their initiation capacity, providing the first example of site-specific interactions that modulate the initiation of DNA replication. The approach exemplified in our studies can pave a path towards a complete understanding of the interactions that spatially and temporally orchestrate chromosome duplication. Our current studies focus on two sets of protein-DNA interactions at replication origins. First, we found that the RepID protein, a member of the DDB1-Cul4-associated-factor (DCAF) protein family, preferentially interacts with replication origins. We have shown that RepID is required for initiation of DNA replication at the origins that bind it. We have further shown that RepID controls the replication program by recruiting the ubiquitin ligase complex, CRL4, to chromatin. In turn, RepID-recruited CRL4 prevents aberrant chromosome re-replication, ensuring that genome duplication occurs only once per cell division. We also discovered another function of RepID and CRL4 in regulating the metaphase-anaphase transition during mitosis. These observations have provided new insights into the mechanisms by which the fidelity of chromosome duplication and segregation can be compromised in cancer cells. Our current studies probe into the question of how cells orchestrate the activity of RepID and CRL4 with other ubiquitin ligases on chromatin to regulate cell proliferation, and characterize in detail the consequences of dysregulation of CRL4 chromatin recruitment and activity at replication origins. Given the mounting evidence that chromosomal re-replication and mis-segregation can be triggered by oncogenes at the onset of tumorigenesis, and the recent development of CRL4 inhibitors (e.g. NEDDylation inhibitors) as anti-cancer therapeutics, these studies have potential translational implications. Second, we identified an interaction between replication origins and the NAD+-dependent protein deacylase SIRT1. Unlike RepID, SIRT1 is not required for DNA replication, but instead, restricts the initiation of DNA replication to a particular group of origins ("baseline" origins) while preventing replication from initiating at other ("dormant") origins. This observation points to a mechanistic link between cellular energy metabolism, epigenetic marks and the regulation of replication origin activation, which play critical roles in maintaining genome integrity. Using SIRT1 activity as a molecular switch to turn dormant origins on and off, we have begun to characterize origin dormancy in detail, mapping the locations of dormant origins and identifying chromatin modifications that distinguish dormant from baseline origins. This work led us to identify components of a signaling network, involving the ATR kinase and the replication accessory protein TOPBP1, that relieves SIRT1-mediated origin dormancy when the baseline origins are stalled, as it occurs when cells are under replication stress. Pointing to the critical role in SIRT1 in maintaining origin dormancy and genome stability, cells with activated dormant origins harbor extrachromosomal elements and exhibit DNA breaks. Our current studies are focused on proteins that associate with dormant origins, the mechanism(s) by which SIRT1 suppresses initiation. We also study molecular pathways, including the abovementioned ATR pathway, that counter SIRT1-mediated suppression to activate dormant origins in cells exposed to stressful conditions. Because ATR inhibitors are explored, at the DTB and elsewhere, as promising therapy agents, these analyses also have a translational potential. The responses of the replication machinery to perturbations are pertinent to human health and the specific cell-cycle regulatory deficiencies in distinct cancer cell types are likely to provide clues to their sensitivity to therapy. Our studies demonstrate that deregulation of the early stages of DNA replication leads to excess replication and subsequent genomic instability through two distinct paths: one that involves activation of dormant origins, and another involving over-activation of baseline origins. We are currently characterizing genetic and epigenetic properties of the replication origins activated by each pathway, as well as protein-DNA interactions modulated by each pathway. Using a combination of single-fiber analyses and sequencing-based techniques, we analyze replication dynamics following exposure to chemotherapeutic agents and chromatin modulators. These studies are expected to identify chromatin targets that are normally involved in preventing excess replication and uncover signaling pathways that convey metabolic status to chromatin. As we learn more about local and distal interactions that promote DNA replication, we will explore pathways that signal back from chromatin to the cell cycle machinery to affect the replication landscape and the cellular responses to anticancer therapy. Our studies rely on tools we have developed to map replication initiation sites throughout the genome and compare replication initiation sites with distinct chromatin features. We are also collaborating with other investigators, within and outside NCI, to characterize genetic and epigenetic features of cancer cells and participate in collaborative efforts that link genetic and epigenetic signatures with responses to therapy. In future studies, we plan to further dissect the molecular interactions that regulate chromosome duplication. Specifically, using a combination of single-fiber analyses, biochemical and computational tools, we will systematically characterize protein-DNA and protein-protein interactions that mediate the effects of RepID, SIRT1, and DNA damage signaling pathways on the chromosome replication program.