Goal:  Our broad goal is to understand the cellular networks that signal to and from chromatin to modulate chromosome duplication. Since many regulatory feedback pathways are deregulated in cancer cells, the results of these studies will help our understanding of cancer biology and elucidate how both normal and cancer cells control their growth.

The challenge of understanding DNA replication:  Loss of genetic control of DNA replication is a hallmark of cancer. Hence, pathways that modulate DNA synthesis can provide good targets for synthetic lethality approaches that specifically target cancer cells. On the other hand, DNA replication problems that go undetected and are not repaired can hamper genomic integrity, eventually resulting in cancer drug resistance. Hence, many anti-cancer drugs target protein complexes involved in DNA replication and the effectiveness of such drugs critically depends on the nature of the genomic lesions affected in particular cancers.

Eukaryotic cells start DNA synthesis at multiple sites on each chromosome, termed replication origins. Replication initiation events in mitotic cells proceed in a precise order and are strictly controlled by a series of cell cycle checkpoint signaling pathways (Aladjem and Redon, Nature Reviews Genetics 2017). These regulatory constraints, however, are often relaxed in cancer. 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, however, because the proteins that form the replication machinery seem to bind to DNA indiscriminately. To gain a complete understanding of the chromosome duplication process we must resolve how these apparently non-specific DNA binding interactions between replication proteins and chromatin translate into highly coordinated replication.

Research strategy:  To gain a better understanding of cell growth regulation, we identify signaling pathways that determine if and when replication would start in particular cells under distinct conditions. We study how replication patterns respond to alterations in gene expression, chromatin modifications and drugs that perturb replication. Because currently identified enzymatic complexes that drive DNA replication do not interact with specific DNA sequences, we are looking for other, sequence-specific protein-DNA interactions that modulate replication at subgroups of replication origins.

For example, we look for proteins that specifically bind replication origins that are active in distinct cells, or origins that start replication at a particular time point within the cell cycle. Once we identify proteins or protein complexes that bind specific groups of replication origins, we ask if those proteins modulate the local activity of the replication machinery. To characterize replication dynamics in cancer cells, we use whole-genome sequencing methods combined with imaging-based single-fiber analyses to characterize the effects of specific proteins on genome duplication patterns.

To provide clues for effective interventions of DNA synthesis pathways, we are also involved in collaborative studies with our colleagues at the NCI’s Developmental Therapeutics Branch.  These studies are aimed at developing better ways to describe regulatory feedback networks that modulate cell cycle progression and characterize the response of cancer cells to anti-tumor therapy.

Protein-DNA interactions at replication origins:  To study DNA-protein interactions that affect DNA replication, we first needed to identify distinct DNA sequences, termed replicators, which facilitate the initiation of DNA replication (Aladjem, Science 1998).  We then used these replicators as baits to identify protein complexes that potentially regulate replication. One of the critical proteins we have identified is RepID, a protein that binds to a group of replication origins (Zhang, Nat Commun. 2016). 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 (Jang, Nat Commun. 2018). These studies provide the first example of a DNA sequence-specific interaction that modulates the initiation of DNA replication. RepID-recruited CRL4 plays a separate, yet essential role for proper chromosome segregation during cell division (Jang, Nat Commun. 2020). Importantly, ReID-recruited CRL4 is involved in a strong inhibitory interaction that normally prevents excess DNA synthesis. If excess DNA synthesis is not inhibited, a part of the genome re-replicates prior to cell division (Fu, Nat Commun. 2021). The resulting excess DNA synthesis is prevalent in cancer cells and can be exploited for therapeutic purposes (Thakur, Trends Genet. 2022). We currently characterize the interactions of RepID with epigenetic modifiers and the replication machinery during normal and disrupted growth.

We have also identified another replication origin binding protein, a phosphorylated form of the NAD+-dependent deacetylase SIRT1 (Utani, Nucleic Acids Res. 2017; Thakur, Nucleic Acids Res. 2022). SIRT1 prevents replication from a group of potential origins ("dormant origins"), hence reducing the probability of transcription-replication collisions, which occur frequently during chronological aging. We are currently investigating how SIRT1 modulates replication origin activation in cells exposed to stressful conditions.

Whole-genome chromatin patterns at replication origins: We combined DNA sequencing with single-DNA-fiber imaging to develop tools that map replication initiation sites and align them with protein binding sites and with epigenetic features throughout the genome. Using these tools, we have generated a comprehensive dataset of replication initiation sites (Martin, Genome Res. 2010; Smith, Epigenetics and Chromatin 2016), demonstrated that replication origin usage is modulated by specific histone modifications (Fu, PLoS Genet 2013) and varies with tissue type (Smith, Epigenetics and Chromatin 2016). To facilitate these studies, we are continuously developing bioinformatics tools (e.g. BAMScale; https://github.com/ncbi/BAMscale) to help decipher the relationships among RepID binding sites and epigenetic features. We are making these tools available to the community to support bioinformatic characterization of DNA-protein interaction loci.

Modulation of DNA replication: An important aspect of our work pertinent to human health is the response of the replication machinery to perturbations. Because an 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 determined the role of a DNA repair endonuclease, Mus81, in modulating the pace of DNA replication (Fu, Nat Commun. 2015) and demonstrated that deregulation of the early stages of DNA replication can proceed in two distinct paths, one involving the activation of dormant origins and the other utilizing the same origins that are used during unperturbed replication (Fu, Nat Commun. 2021).  We are currently studying how replication origin binding proteins (e.g., SIRT1) affect the cellular response to perturbation of cell cycle progression.

Literature cited:

Aladjem MI, Rodewald LW, Kolman JL and Wahl GM. Genetic dissection of a mammalian replicator in the human beta-Globin locus. Science 281:1005-1009, 1998.

Aladjem MI, Redon CE. Order from clutter: selective interactions at mammalian replication origins. Nat Rev Genet. 18:101-116, 2017.

Fu H, Martin MM, Regairaz M, Huang L, You Y, Lin CM, Ryan M, Kim R, Shimura T, Pommier Y, Aladjem MI. The DNA repair endonuclease Mus81 facilitates fast DNA replication in the absence of exogenous damage. Nat Commun. 6:6746, 2015.

Fu H, Maunakea AK, Martin MM, Huang L, Zhang Y, Ryan M, Kim R, Lin CM, Zhao K, Aladjem MI. Methylation of histone H3 on lysine 79 associates with a group of replication origins and helps limit DNA replication once per cell cycle. PLoS Genet. 9:e1003542, 2013.

Fu H, Redon CE, Thakur BL, Utani K, Sebastian R, Jang SM, Gross JM, Mosavarpour S, Marks AB, Zhuang SZ, Lazar SB, Rao M, Mencer ST, Baris AM, Pongor LS, Aladjem MI. Dynamics of replication origin over-activation. Nat Commun. 12:3448. 2021.

Jang SM, Nathans JEF, Fu H, Redon CE, Jenkins LM, Thakur  B, PongorLS, Baris AM, GrossJM, O’Neill M, IndigFE, Cappell S, Aladjem MI. The RepID-CRL4 ubiquitin ligase complex regulates metaphase to anaphase transition via BUB3 degradation. Nature Communications 11, 24. 2020.

Jang SM, Zhang Y, Utani K, Fu H, Redon CE, Marks AB, Smith OK, Redmond CJ, Baris AM, Tulchinsky DA, Aladjem MI. The replication initiation determinant protein (RepID) modulates replication by recruiting CUL4 to chromatin. Nat Commun. 9:2782, 2018.

Kim R, Smith OK, Wong WC, Ryan AM, Ryan MC, Aladjem MI. ColoWeb: A Resource for Analysis of Colocalization of Genomic Features. BMC Genomics 16:142, 2015.

Martin MM, Ryan M, Kim R, Zakas AL, Fu H, Lin CM, Reinhold WC, Davis SR, Bilke S, Liu H, Doroshow JH, Reimers MA, Valenzuela MS, Pommier Y, Meltzer PS, Aladjem MI. Genome-wide depletion of replication initiation events in highly transcribed regions. Genome Res. 21:1822-1832, 2011.

Pongor LS, Gross JM, Vera Alvarez R, Murai J, Jang SM, Zhang H, Redon C, Fu H, Huang SY, Thakur B, Baris A, Marino-Ramirez L, Landsman D, Aladjem MI*, Pommier Y. BAMscale: quantification of next-generation sequencing peaks and generation of scaled coverage tracks. Epigenetics and Chromatin. 13:21. 2020.

Smith OK, Kim RG, Fu H, Martin M, Utani K, Zhang Y, Marks AB, Lalande M, Chamberlaine S, Libbrecht MW, Bouhassira EE, Ryan MC, Noble WC, Aladjem MI. Distinct Epigenetic Features of Differentiation-Regulated Replication Origins. Epigenetics and Chromatin 9:18. 2016.

Thakur BL, Ray A, Redon CE, Aladjem MI.  Preventing excess replication origin activation to ensure genome stability. Trends Genet. 38:169-181. 2022.

Thakur BL, Baris AM, Fu H, Redon CE, Pongor LS, Mosavarpour S, Gross JM, Jang SM, Sebastian R, Utani K, Jenkins LM, Indig FE, Aladjem MI. Convergence of SIRT1 and ATR signaling to modulate replication origin dormancy. Nucleic Acids Res. 50:5111-5128. 2022.

Utani K, Fu H, Jang SM, Marks AB, Smith OK, Zhang Y, Redon CE, Shimizu N, Aladjem MI. Phosphorylated SIRT1 associates with replication origins to prevent excess replication initiation and preserve genomic stability. Nucleic Acids Res. 45:7807-7824, 2017.

Zhang Y, Huang L, Fu H, Smith OK, Lin CM, Utani K, Rao M, Reinhold WC, Redon CE, Ryan M, Kim RG, You Y, Hanna H, Boisclair Y, Long Q, Aladjem  MI. A Replicator-Specific Binding Protein Essential for Site-Specific Initiation of DNA Replication in Mammalian Cells. Nat Commun. 7:11748, 2016.