Loss of genetic control causing inappropriate and excessive proliferation is a hallmark of cancer cells. Many of the defective regulatory pathways that lead to aberrant cell growth converge on molecular events that facilitate DNA replication. Replication regulatory pathways can provide good targets for synthetic lethality approaches that specifically kill cancer cells, but replication problems that go undetected can affect genomic integrity, triggering genomic instability that eventually might result in cancer drug resistance. Hence, many anti-cancer drugs target various aspects of DNA replication and the effectiveness of such drugs critically depends on the nature of the lesions affected in particular cancers. The DNA replication group at LMP aims to gain a detailed understanding of the early stages of the replication process in the context of chromatin and cell cycle signaling and investigate how the process is controlled during normal and perturbed growth in cancer and non-cancer cells. Eukaryotic cells start chromosomal replication from multiple regions (replication origins) on each chromosome to duplicate the entire genome prior to each cell division. Replication initiation events occur in precise order during the S-phase of the cell cycle and are subject to strict regulatory processes that are often dysregualted in cancer. Studies in the DNA replication group have previously revealed some of the principles by which the location and the timing of replication initiation events are determined and started to decipher how replication is linked to distinct chromatin modifications. Current studies aim to identify components of the cell cycle signaling network that interact with proteins found on replication origins during normal and perturbed growth and ask how DNA replication coordinates with other chromatin transactions such as transcription, DNA repair and chromosome condensation. To that end, we take two complementary approaches. First, we use biochemical and genetic approaches to identify and characterize DNA sequences that facilitate replication and then use those sequences as baits for proteins that bind such sequences to regulate DNA replication. Second, we use massively parallel sequencing and replication imaging methodology to study the genome-wide dynamics of DNA replication and determine how replication patterns respond to alterations in gene expression, chromatin modifications and drugs that perturb replication. For the first approach, we study DNA sequences (termed replicators) that facilitate initiation of DNA replication at their endogenous chromosomal sites or when they are removed from their endogenous location and transferred to ectopic chromosomal sites. In previous studies, we have identified replicator sequences in mammalian cells and dissected the genetic determinants of replicator activity in two such replicators (Aladjem, Rodewald et al. 1998;Wang, Lin et al. 2004;Wang, Lin et al. 2006). We have observed that not all potential replicators initiate replication during each cell cycle and that epigenetic processes and distal chromatin interactions play a role in determining if and when a particular replicator will be used during each S-phase ((Fu, Wang et al. 2006) reviewed in (Aladjem 2007) and in (Conner and Aladjem 2012)) . Recently, we used the replicator we have identified (RepP) as a bait to identify two discrete DNA-protein complexes. One RepP-associated complex includes chromatin-remodeling proteins and affects replication timing and transcriptional activity (Huang, Fu et al. 2011). The other complex is required for DNA replication (Huang et al., submitted) and includes a novel protein termed Rep-ID. We have characterized Rep-ID interaction with chromatin to reveal that Rep-ID binds chromatin in early G1 phase and dissociates from chromatin as DNA replication proceeds. Importantly, Rep-ID deficient cells exhibit fewer replication initiation sites and aberrant replication, suggesting that Rep-ID is an important determinant of replication initiation events. Next, we will ask how Rep-ID interactions are altered as cells progress through the cell cycle and whether those chromatin interactions would change in normal and cancer cells exposed to environmental challenges and anti-cancer drugs. For the second approach, we have developed tools for mapping replication initiation sites on a genome scale and mining those data and correlate with other chromatin features. Last year we have published the first comprehensive mapping of replication initiation sites in mammalian cells (Martin, Ryan et al. 2011). The dataset created by these studies encompasses the locations of replication initiation sites throughout the entire non-repetitive genomes of the analyzed transformed and non-transformed cells. Combined with the first approach, we have used massively parallel sequencing to analyze the binding patterns of Rep-ID. These studies revealed that Rep-ID preferentially associates with replication origins and the likelihood of binding to Rep-ID decreases with the distance from replication initiation sites and demonstrated that this novel protein binds a subset of replication initiation sites (Huang et al., submitted). We have also collaborated with Keji Zhao (NHLBI) to identify a chromatin modification, trimethylation of histone H3 on lysine 79, that associates with replication initiation sites and prevents over-replication (Fu et al., submitted). Our studies revealed that the decision when and where DNA would replicate on chromatin depends not only on DNA-protein interactions at the local level but also on interactions at a distance. Our recent findings suggest that at the human beta globin locus Rep-ID participates in a complex between the replicator and the locus control region, which is essential for initiation of DNA replication (Huang et al., submitted). This observation proposes a hypothesis for the mechanism underlying the role of distal interactions in regulating DNA replication in metazoan loci. We anticipate that Rep-ID is the first of a family of proteins that recruit the replication machinery to specific replicators and modulate DNA replication in metazoans. Finding replicator-binding proteins that determine whether initiation occurs at particular sites is critical to the understanding of how cell cycle regulatory network interact with chromatin to start replication. To investigate how cells respond to perturbed replication, we utilized single fiber analyses of DNA replication to identify a new pathway involved in the cellular response to replicative stress. We have shown previously that low non-toxic doses of replication inhibitors decelerate replication by a mechanism involving the cancer-predisposing protein BLM helicase, Mus81 nuclease and ATR kinase (Shimura, Martin et al. 2007;Shimura, Torres et al. 2008). Currently we use a mutated Mus81 protein that does not contain an active exonuclease to ask whether the exonuclease activity of Mus81 participates in the regulating the pace of DNA replication and in the response of cells to perturbation of the replication program by anti-cancer drugs. We have expanded the replication initiation site dataset to include several cancer cell lines and cells in which the exonuclease activity of Mus81 was compromised (Fu et al., submitted). The combination of genome-scale sequencing of replication initiation sites and single fiber analyses provide important insights into the organization of replication initiation events and the cellular responses to signals that might perturb DNA replication. Future studies will reveal how the replication landscape would change in normal and cancer cells exposed to environmental challenges and anti-cancer drugs.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Investigator-Initiated Intramural Research Projects (ZIA)
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National Cancer Institute Division of Basic Sciences
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Aladjem, Mirit I; Redon, Christophe E (2017) Order from clutter: selective interactions at mammalian replication origins. Nat Rev Genet 18:101-116
Utani, Koichi; Fu, Haiqing; Jang, Sang-Min et al. (2017) Phosphorylated SIRT1 associates with replication origins to prevent excess replication initiation and preserve genomic stability. Nucleic Acids Res 45:7807-7824
Wang, Yating; Khan, Abid; Marks, Anna B et al. (2017) Temporal association of ORCA/LRWD1 to late-firing origins during G1 dictates heterochromatin replication and organization. Nucleic Acids Res 45:2490-2502
Smith, Owen K; Kim, RyanGuk; Fu, Haiqing et al. (2016) Distinct epigenetic features of differentiation-regulated replication origins. Epigenetics Chromatin 9:18
Zhang, Ya; Huang, Liang; Fu, Haiqing et al. (2016) A replicator-specific binding protein essential for site-specific initiation of DNA replication in mammalian cells. Nat Commun 7:11748
Marks, Anna B; Smith, Owen K; Aladjem, Mirit I (2016) Replication origins: determinants or consequences of nuclear organization? Curr Opin Genet Dev 37:67-75
Fu, Haiqing; Martin, Melvenia M; Regairaz, Marie et al. (2015) The DNA repair endonuclease Mus81 facilitates fast DNA replication in the absence of exogenous damage. Nat Commun 6:6746
Luna, Augustin; McFadden, Geoffrey B; Aladjem, Mirit I et al. (2015) Predicted Role of NAD Utilization in the Control of Circadian Rhythms during DNA Damage Response. PLoS Comput Biol 11:e1004144
Kim, RyangGuk; Smith, Owen K; Wong, Wing Chung et al. (2015) ColoWeb: a resource for analysis of colocalization of genomic features. BMC Genomics 16:142
Bartholdy, Boris; Mukhopadhyay, Rituparna; Lajugie, Julien et al. (2015) Allele-specific analysis of DNA replication origins in mammalian cells. Nat Commun 6:7051

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