Our long term goals include elucidating several fundamental and long standing questions in mammalian chromosome replication. These include: 1) Are there genetically defined sequences (replicators) that determine sites for initiation of DNA replication irrespective of whether initiation occurs at specific sites or in delocalized regions? 2) What is the significance and mechanism of pausing of replication forks at specific sites? 3) Is there a role for site-specific termination of replication in mammalian chromosomes? 4) What determines the precise temporal replication program of specific chromosomal loci during the S phase? We have recently made the important observation that latent Epstein-Barr virus (EBV) replication resembles, in several ways, chromosomal replication in human cells. EBV is an oncogenic human herpesvirus that can cause infectious mononucleosis and is closely associated with African Burkitt's lymphoma and nasopharyngeal carcinoma, one of the most common forms of cancer in Asia. In individuals infected with HIV or in immunosuppressed transplantation patients, EBV-associated lymphoproliferative disorders can occur at high frequency. The EBV genome persists extrachromosomally in a latent state in a small number of B lymphocytes in most humans and is maintained throughout their entire lifetime. EBV episomes are duplicated by the host cell machinery precisely once per cell cycle. An understanding of the mechanisms by which EBV replicates and is maintained should provide valuable insights into this important human pathogen. In this proposal we will use EBV as a model system to answer several fundamental questions about mammalian DNA replication.
Specific aims i nclude: 1) We will identify potential cis- acting sequences that establish the delocalized initiation region that we have recently described in EBV. To accomplish this we will first generate recombinant EBV genomes with mutations in the delocalized initiation region. We will then examine these modified EBV strains by two- dimensional gel electrophoresis to identify sequences important for delocalized initiation. 2) We will determine the cis- and trans-acting requirements for the pausing of replication forks in the EBV genome. We will first identify the precise location where forks are stalled and then specifically mutagenize DNA sequences to identify the critical elements required.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Molecular Cytology Study Section (CTY)
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Albert Einstein College of Medicine
Anatomy/Cell Biology
Schools of Medicine
United States
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Pan, Xiaolei; Drosopoulos, William C; Sethi, Louisa et al. (2017) FANCM, BRCA1, and BLM cooperatively resolve the replication stress at the ALT telomeres. Proc Natl Acad Sci U S A 114:E5940-E5949
Madireddy, Advaitha; Purushothaman, Pravinkumar; Loosbroock, Christopher P et al. (2016) G-quadruplex-interacting compounds alter latent DNA replication and episomal persistence of KSHV. Nucleic Acids Res 44:3675-94
Madireddy, Advaitha; Kosiyatrakul, Settapong T; Boisvert, Rebecca A et al. (2016) FANCD2 Facilitates Replication through Common Fragile Sites. Mol Cell 64:388-404
Calderano, Simone Guedes; Drosopoulos, William C; Quaresma, Marina Mônaco et al. (2015) Single molecule analysis of Trypanosoma brucei DNA replication dynamics. Nucleic Acids Res 43:2655-65
Drosopoulos, William C; Kosiyatrakul, Settapong T; Schildkraut, Carl L (2015) BLM helicase facilitates telomere replication during leading strand synthesis of telomeres. J Cell Biol 210:191-208
Gerhardt, Jeannine; Zaninovic, Nikica; Zhan, Qiansheng et al. (2014) Cis-acting DNA sequence at a replication origin promotes repeat expansion to fragile X full mutation. J Cell Biol 206:599-607
Gerhardt, Jeannine; Tomishima, Mark J; Zaninovic, Nikica et al. (2014) The DNA replication program is altered at the FMR1 locus in fragile X embryonic stem cells. Mol Cell 53:19-31
Murphy, Anar K; Fitzgerald, Michael; Ro, Teresa et al. (2014) Phosphorylated RPA recruits PALB2 to stalled DNA replication forks to facilitate fork recovery. J Cell Biol 206:493-507
Jeong, Yeon-Tae; Rossi, Mario; Cermak, Lukas et al. (2013) FBH1 promotes DNA double-strand breakage and apoptosis in response to DNA replication stress. J Cell Biol 200:141-9
Drosopoulos, William C; Kosiyatrakul, Settapong T; Yan, Zi et al. (2012) Human telomeres replicate using chromosome-specific, rather than universal, replication programs. J Cell Biol 197:253-66

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