Initiation of DNA Replication in Mammalian Cells

Division Of Basic Sciences - Nci

Investigators

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Abstract

Our studies focus on cellular signaling pathways that regulate cell proliferation, focusing on the location, timing and progression of DNA synthesis in cancer cells. Since many regulatory pathways that affect chromosome duplication are deregulated in cancer, our findings can facilitate understanding the regulation of cancer cell proliferation and the determinants of response to chemotherapeutic drugs. Our current research plan is built on our previous findings, which include whole-genome-scale mapping of replication initiation sites (replication origins) in human cells, identification of chromatin modifications and protein complexes that selectively assemble on origins and modify replication initiation patterns, and characterization of signaling pathways that detect and respond to disruptions of DNA synthesis. This year we report two major discoveries. First, we identified a group of proteins, termed mediators of replication and DSBs (MRDs), which facilitate a local, chromatin-confined genome maintenance mechanism that inhibits replication initiation in DNA breaks-containing topologically associating domains (TADs) without effecting DNA synthesis at other genomic locations (Sebastian et al., Nature, 2025). Dysregulation of MRDs, or disruption of 3D chromatin architecture by dissolving TADs, results in inadvertent replication within damaged chromatin and increased DNA damage in cancer cells. In addition to subunits of the cohesin complex, MRDs include chromatin modulators such as 53BP1 and Rif1 as well as the TIMELESS-TIPIN complex and the WEE1 kinase, which actively dislodges the TIMELESS-TIPIN complex from replication origins adjacent to DNA breaks and prevents initiation of DNA synthesis. Second, we report that the recovery from perturbation of the DNA replication program involves the redistribution of two origin-binding proteins, RecQL4 and MTBP, to replication origins (dormant origins). pRecQL4-mediated MTBP redistribution activates dormant origins and facilitates backup replication upon recovery from replication stress (Thakur et al., Nature Communications, 2025). These studies build upon our previous finding that SIRT1, a protein deacetylase involved in metabolic interactions and implicated in modulating the aging process, acts as a molecular switch that suppresses replication initiation at dormant origins. When SIRT1 is active, baseline origins initiate replication, whereas dormant origins remain inactive and unless replication stalls or slows (replication stress). Consequently, SIRT1 activity is critical to prevent excess replication, resulting in collisions between the transcription and replication machineries (R-loops). The decline in SIRT1 activity during chronological aging may underlie the aging-associated increased prevalence of R-loops. As baseline and dormant origins bind apparently identical pre-replication complexes, using SIRT1 as a molecular switch allowed us to identify MTBP as a differential origin activator during normal proliferation and in response to replication stress, and pinpoint RecQL4 as a determinant of origin dormancy. We are now exploring how the phosphorylation of RecQL4, a RecQ helicase mutated in a series of devastating DNA repair syndromes associated with cancer susceptibility, forms the mechanistic basis for dormant origins suppression and prevention of excess DNA synthesis. In addition, we continue to investigate the involvement of ubiquitin ligases in preventing excess DNA synthesis. We have previously shown that RepID, a DCAF component of the CRL4 ubiquitin ligase complex, prevents partial genome re-replication by recruiting CRL4 to chromatin. We are currently mapping the chromatin targets of RepID and investigating the downstream effects of its depletion on replication initiation profiles. We also collaborate with other investigators to characterize replication profiles of extrachromosomal DNA in cancer cells. In another collaboration, we explore if potential modulation of ubiquitin ligases to induce excess replication could be used as a synergistic approaches for therapeutic intervention in cancers that exhibit high levels of replication stress. Our studies are facilitated by novel bioinformatics and biochemical approaches that we developed and implemented. Our imaging-based assays for assessing the effects of DNA breaks on DNA replication were instrumental in the discovery of the MRD pathway. We are currently developing computational approaches for interpreting single fiber analyses of DNA replication. We plan to leverage our knowledge of molecular interactions at replication origins and chromatin structure to reveal a previously unknown vulnerability in the DNA replication machinery that may be exploited to therapeutically target cancer cells.