For several years we have studied replication initiation in specific DNA sequences. Our development of Single Molecule Analysis of Replicated DNA (SMARD) has allowed us to study the replication of segments encompassing up to 1 Mb of a genomic locus. In the present proposal we will use this powerful approach to examine replication in three model systems: the Igh locus, human and mouse telomeres, and the triplet repeat- containing Fragile X Mental Retardation (FMR1) locus. We have shown that origins are absent in a region of the IgH-constant region locus in non-B cells but are activated at preferred sites in pro-B cells. We will use Igh- C locus primarily as a model to examine responses to replication stress, including activation of latent origins. We will determine pre-replication complex (pre-RC) localization and nucleosome organization in the Igh-C locus by ChIP. Comparison of SMARD origin mapping with pre-RC occupancy and nucleosome landscape will provide insights into the mechanisms of latent origin activation. In the second model we have made essential technical advances in SMARD that allow us to study replication in specific telomere loci and have obtained detailed information on telomere replication programs. A key finding is that human and mouse telomeres appear to be replicated by chromosome-specific replication programs rather than a universal program. We will use SMARD to determine how these programs are affected by replication stress and what aspects of replication are challenged by telomeres. We will also examine telomere dysfunction at specific telomeres. These analyses will reveal the flexibility of telomere replication programs, including the ability to activate latent origins, and establish if specific replication programs render telomeres more susceptible to stress and dysfunction. We also have evidence to suggest that BLM helicase directly contributes to efficient telomere replication. We will use SMARD to determine how BLM facilitates telomere replication and if telomeres are more reliant on BLM than internal genomic regions. We will also determine the functional overlap between BLM and other helicases in telomere replication. In the third model we will study trinucleotide repeat (TNR) expansion, which is associated with more than 30 inherited diseases including Fragile X syndrome (FXS). Many model systems have been generated to study the role of replication in TNR expansion, but models of expansion at endogenous human loci are lacking. Using SMARD we are now able to compare the role of replication initiation sites in TNR expansion in the endogenous FMR1 locus (the affected locus in FXS) in human cells, including cell lines from women at risk for giving birth to FXS males, that have and do not have TNR expansion to elucidate the role of origin site selection on TNR instability. We will also determine the contribution of CTCF binding to FMR1 replication programs. Based on our preliminary data, we expect these proposed studies will both greatly increase our understanding of telomere and TNR replication and allow us to establish new paradigms for the role replication origin selection in health and disease.

Public Health Relevance

Unusual and complex regions in chromosomes challenge faithful replication of chromosomes. Errors in replication of these regions can lead to many diseases, including cancer and neurological disorders such as Fragile X syndrome. Understanding how unusual and complex regions are replicated both normally and under stress should have important implications for normal development, predisposition to disease and the management of therapies such as the use of many cancer drugs that target the process of DNA replication.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Molecular Genetics B Study Section (MGB)
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Reddy, Michael K
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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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