Repetitive DNA sequences often challenge DNA replication, which can lead to genomic instability. Aberrant replication of two repeat-rich regions in particular, telomeres and common fragile sites (CFS), have pathological consequences. The goal of this proposal is to elucidate mechanisms that ensure normal telomere and CFS replication and protect genomic integrity. Replication fork stalling at repetitive sequences is thought to be a main causative factor underlying both telomere- and CFS-associated genomic instability. Using our single molecule approach, termed SMARD (single molecule analysis of replicated DNA), we have shown that stalling occurs at both telomeres and CFS. Therefore we propose to examine how stalling is overcome at these important chromosomal elements with the following aims.
In Aim1, we will establish the role of replication initiation within telomeres in resuming stalled replication and maintaining proper telomere function. Initiation of replication ahead of a stalled fork is a key replication recovery response. We will disrupt replication fork progression in human and mouse cells using replication inhibitors, G-quadruplex stabilizers and replication barriers, and examine telomere replication to establish that telomeric initiation will occur to rescue stalled replication. Key events driving telomere-associated genetic instability are telomere shortening and failure to suppress DNA damage responses and deleterious repair (telomere dysfunction). We will determine if reduced or compromised telomeric initiation leads to defective telomere replication resulting in telomere loss and dysfunction, to establish whether telomeric initiation provides a protective mechanism for maintaining proper telomere function.
In Aim 2, we will establish mechanism(s) that facilitate the replication of stall-prone regions in CFS loci. Our preliminary studies reveal that the Fanconi anemia complementation group D2 protein (FANCD2) plays an important role in facilitating CFS replication by alleviating replication pausing. Thus, we will perform studies to further define its role in CFS replication. Since FANCD2 interacts with translesion polymerases, which have been implicated in CFS replication, we will establish whether FANCD2 recruits these polymerases to aid in CFS replication. We will also investigate whether FANCD2 alleviates transcription- associated obstacles to replication fork progression including RNA: DNA hybrids, because FANCD2?deficient cells display an increased abundance these hybrids genome-wide. Our preliminary studies reveal that changes in replication origin usage are associated with FANCD2 deficiency. Because altered origin usage is linked to changes in chromosome organization, we will determine if FANCD2 promotes a chromosome architecture that facilitates accurate CFS replication. We expect that these proposed studies will both greatly increase our understanding of telomere and CFS replication and allow us to establish new paradigms for replication of repetitive elements and their role in disease etiology.
Faithful chromosome replication is essential to maintain normal cell function. Our goal is to elucidate how two difficult to replicate chromosomal elements, telomeres and common fragile sites, are accurately replicated to prevent genetic dysfunction. Understanding the mechanisms that ensure proper replication of these biologically important chromosomal elements will provide needed insight into to the causes of cancer, and to congenital and age-related degenerative diseases.
|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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