The DNA Replication Group aims to understand how information from the cell cycle machinery leads to the initiation of DNA replication. Proper cell growth depends on a network of interacting molecules that prevents DNA replication and cell division under unfavorable conditions. Disruptions in the intricate balance between components of this network may lead to cancer;however, interfering with signals transmitted by the cell cycle signaling network is an important tool for cancer therapy. A better understanding of the cell cycle is fundamental to the development of rational, knowledge-based strategies to combat cancer and utilize stem cells to improve human health. To study cell cycle signaling at the chromatin level, we specify DNA sequences that determine whether, where, and when replication will occur. DNA sequences that determine the location of replication initiation are called replicators. Replicators are identified by their ability to start replication when transferred from their original genomic locus to ectopic genomic sites [Aladjem, MI, et al. Science 281: 1005-9, 1998]. Genetic dissection of replicators (see Specific Aim 1) delineates the sequence requirements for starting DNA replication. This year we have started a detailed analysis of DNA-protein interactions of these sequences. We also report (Specific Aim 2) that the timing of DNA replication during the S-phase of the cell cycle can be altered [Lin CM, et al. Curr Biol 13: 1019-28, 2003]. We now alter replication timing as a tool to elucidate genetic and epigenetic factors that determine replication timing. We have recently started to use single molecule analyses of DNA replication to determine replication timing of particular sequences (Specific Aim 2) and to evaluate the effect of changes in metabolic conditions and exposure to anti-cancer drugs on initiation patterns [Shimura T, et al. J Mol Biol 367: 665-80, 2007; 375:1152-64m 2008] (Specific Aim 3). The studies outlined above provide insights into the interactions of the cell cycle machinery with chromatin to control DNA replication during normal growth and in response to replication-perturbing drugs. Below is a summary of recent findings, summarized briefly for each specific aim. 1) Characterization of replicators, genetic elements that affect the location of replication initiation. We have established that the replication initiation region within the human beta-globin locus contains two independent, non-overlapping replicators and have identified sequence motifs that are required for initiation of DNA replication within these replicators [Wang L, et al. Mol Cell Biol 24, 3373-86, 2004]. At the beta-globin locus, those sequence motifs interact with each other to determine the location of replication initiation events, implying a modular structure for mammalian replicators [Wang L, et al. Hum Mol Genet 15: 2613-22, 2006]. The above studies are in line with the emerging understanding that replication patterns in metazoans are dynamically regulated by a combination of sequence and epigenetic modifications [Aladjem MI. Nat Rev Genet 8: 588-600, 2007]. Our recent observations suggest that replicators bind particular proteins at specific stages of the cell cycle. Our current efforts include identification and characterization of replicator-binding proteins and protein complexes and elucidation of the role of such protein-DNA interactions in the early stages of DNA replication. 2) Analysis of the effect of DNA sequence and chromatin structure on replication timing. We have identified DNA sequences that affect the timing of DNA replication [Lin CM, et al. Curr Biol 13: 1019-28, 2003;Feng YQ, et al. Mol Cell Biol 25: 3864-74, 2007] and have shown that the timing of DNA replication correlates with the status of chromatin condensation and with epigenetic factors, such as methylation of CpG sequences [Feng YQ, et al. PLoS Genet 2, e65, 2006] and histone modifications [Lin CM, et al. Curr Biol 13: 1019-28, 2003;Fu H, et al. Nat Biotechnol 24: 572-6, 2006]. Tissue-specific patterns of replication timing can be conserved in evolution even in loci that do not conserve replication initiation patterns [Aladjem MI, et al. Mol Cell Biol 22: 442-5, 2002]. We have shown that functional replicator sequences (but not mutated replicators) prevented gene silencing and replication delay and prohibited chromatin condensation [Fu H, et al. Nat Biotechnol 24: 572-6, 2006]. Ongoing studies focus on the characterization of point mutations in replicator sequences on the timing of DNA replication and on the applicability of our initial findings to the stabilization of gene expression using genes with therapeutic relevance. 3) Identification of cellular signaling interactions induced by the perturbation of DNA replication. We have uncovered a cellular response pathway involving BLM helicase, Mus81 nuclease, ATR kinase and the non-homologous recombination cascade. This pathway responds to perturbation of DNA replication following exposure to mild drug-induced perturbation of DNA replication, which is below the threshold of the cell cycle checkpoint response. Cells that are exposed to mild perturbation of DNA replication exhibit transient DNA breaks that are formed by Mus81 with the cooperation of BLM helicase and ATR kinase [Shimura T, et al. J Mol Biol 375: 1152-64, 2008]. In cells that contain an intact nonhomologous end-joining pathway, these DNA breaks are transient and cells rapidly resume replication in the presence of the inhibitor, albeit at a slow rate. DNA breaks persist in cells that are deficient in components of the nonhomologous end-joining pathway such as DNA-PK and XRCC4;such cells are unable to resume DNA replication and activate a cell cycle checkpoint response after a mild inhibition of DNA synthesis [Shimura T, et al. J Mol Biol 367: 665-80, 2007]. These recent findings propose that replication-induced DNA breaks do not always arise passively from polymerase collisions;breaks can also form as intermediates in the resolution of perturbed replication.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC010411-10
Application #
7965300
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
10
Fiscal Year
2009
Total Cost
$966,780
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
Fu, Haiqing; Baris, Adrian; Aladjem, Mirit I (2018) Replication timing and nuclear structure. Curr Opin Cell Biol 52:43-50
Utani, Koichi; Aladjem, Mirit I (2018) Extra View: Sirt1 Acts As A Gatekeeper Of Replication Initiation To Preserve Genomic Stability. Nucleus 9:261-267
Warburton, Alix; Redmond, Catherine J; Dooley, Katharine E et al. (2018) HPV integration hijacks and multimerizes a cellular enhancer to generate a viral-cellular super-enhancer that drives high viral oncogene expression. PLoS Genet 14:e1007179
Jang, Sang-Min; Redon, Christophe E; Aladjem, Mirit I (2018) Chromatin-Bound Cullin-Ring Ligases: Regulatory Roles in DNA Replication and Potential Targeting for Cancer Therapy. Front Mol Biosci 5:19
Aladjem, Mirit I; Redon, Christophe E (2017) Order from clutter: selective interactions at mammalian replication origins. Nat Rev Genet 18:101-116
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
Marks, Anna B; Fu, Haiqing; Aladjem, Mirit I (2017) Regulation of Replication Origins. Adv Exp Med Biol 1042:43-59
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
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

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