Our long term goal is to understand the control of the determinants of initiation of DNA replication in mammalian cells. The system we have developed for this purpose rests on the identification of the replication initiation sites in the mouse immunoglobulin heavy chain gene (Igh) locus. We have established that the timing of replication at this locus differs radically between cells in the early and late stages of B lineage development. In cells in the early stages of development the entire Igh locus replicates early in the S phase, while in the late stages part of the locus replicates later in S phase. We inferred that the number and location of replication initiation sites changes during B cell development. We propose now to track these changes in cells of the B lineage throughout the course of differentiation. We will also examine the relationship between histone modifications and the activation of new replication initiation sites. We will rely on the new approach that we have developed, as set out in the objectives of our previous application, for the study of DNA replication in single-copy gene loci in mammalian cells; we have termed this Single Molecule Analysis of Replicated DNA (SMARD). This approach has recently enabled us to analyze the replication initiation sites in the single copy Igh locus of mammalian cells. It further affords us now a means of following, through all the stages of B cell development, the replication of the Igh locus in both cell lines and primary cells. The latter have not previously been amenable to study because of the large number of cells in each developmental stage demanded by the conventional, electrophoretic, method of analysis. Both normal and malignant cells will be studied. Our studies will establish whether indeed replication origin usage changes with development and ultimately will make it possible to resolve the question of whether the replication fork direction in the Igh locus governs some B cell specific processes such as VDJ recombination, isotype switching and somatic hypermutation. We will use SMARD to localize and characterize the developmentally regulated replication initiation sites in B cell lines and primary cells representing successive stages of differentiation. We will study DNA replication throughout the Igh locus in order to identify replication initiation sites that are active in pro and pre B cells and silenced in cells representing later stages of B cell development. We will study the acetylation status of histones at activated and silenced developmentally regulated initiation sites in the Igh locus in B cell lines and primary cells. Finally, we will use gene targeting in ES cells to determine the impact of transcription, possibly through the modification of chromatin structure, on the activation of replication origins in primary B lineage lineage cells.

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National Institute of General Medical Sciences (NIGMS)
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Cell Development and Function Integrated Review Group (CDF)
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Dearolf, Charles R
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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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