Control of DNA replication is critical to ensure accurate copy number of genes, because both loss of gene copies and increased copy number by amplification are associated with the onset of cancer and progression to metastatic states. Elucidation of regulatory mechanisms for metazoan DNA replication has been hampered by difficulty in defining specific origins of replication and monitoring their activation. Most animals, including humans, contain polyploid tissues in which the DNA content of the cells is increased by a modified cell cycle, the endo cycle, lacking mitosis. In addition to the overall increase in genomic DNA, in many polyploid cells differential DNA replication occurs in which specific genomic intervals are not replicated or in some cases are over-replicated and amplified. These instances of differential DNA replication provide superb models for elucidating the structure and regulation of metazoan DNA replication origins. Using genomic methodologies, amplified genomic regions were identified in the Drosophila ovarian follicle cells and single copy, euchromatic underreplicated regions were found in the larval salivary gland. The replication origins within the amplified regions are subject to developmental control and permit analysis of the factors responsible for origin activation and repression, as well as discovery of mechanisms by which metazoan DNA replication is initiated.
The aims of this research are to exploit these Drosophila replication models to define the mechanisms that activate initiation at the origins during follicle cell differentiation, to determine how replication origins are inactivated and to use mutants to identify new regulatory proteins, and to analyze mechanisms controlling replication fork progression. The identification of defined replication origins whose duplication can be quantified, the ability to detect replication proteins bound at these origins and moving with the replication forks, combined with a collection of mutants affecting these processes permits these experimental goals to be achieved.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM057960-11
Application #
7845088
Study Section
Molecular Genetics B Study Section (MGB)
Program Officer
Hagan, Ann A
Project Start
1999-05-01
Project End
2012-05-31
Budget Start
2010-06-01
Budget End
2011-05-31
Support Year
11
Fiscal Year
2010
Total Cost
$389,583
Indirect Cost
Name
Whitehead Institute for Biomedical Research
Department
Type
DUNS #
120989983
City
Cambridge
State
MA
Country
United States
Zip Code
02142
Hua, Brian L; Orr-Weaver, Terry L (2017) DNA Replication Control During Drosophila Development: Insights into the Onset of S Phase, Replication Initiation, and Fork Progression. Genetics 207:29-47
Alexander, Jessica L; Beagan, Kelly; Orr-Weaver, Terry L et al. (2016) Multiple mechanisms contribute to double-strand break repair at rereplication forks in Drosophila follicle cells. Proc Natl Acad Sci U S A 113:13809-13814
Alexander, Jessica L; Orr-Weaver, Terry L (2016) Replication fork instability and the consequences of fork collisions from rereplication. Genes Dev 30:2241-2252
Orr-Weaver, Terry L (2015) When bigger is better: the role of polyploidy in organogenesis. Trends Genet 31:307-15
Frawley, Laura E; Orr-Weaver, Terry L (2015) Polyploidy. Curr Biol 25:R353-8
Nordman, Jared T; Orr-Weaver, Terry L (2015) Understanding replication fork progression, stability, and chromosome fragility by exploiting the Suppressor of Underreplication protein. Bioessays 37:856-61
Alexander, Jessica L; Barrasa, M Inmaculada; Orr-Weaver, Terry L (2015) Replication fork progression during re-replication requires the DNA damage checkpoint and double-strand break repair. Curr Biol 25:1654-60
Hua, Brian L; Li, Sharon; Orr-Weaver, Terry L (2014) The role of transcription in the activation of a Drosophila amplification origin. G3 (Bethesda) 4:2403-8
Nordman, Jared T; Kozhevnikova, Elena N; Verrijzer, C Peter et al. (2014) DNA copy-number control through inhibition of replication fork progression. Cell Rep 9:841-9
Sher, Noa; Bell, George W; Li, Sharon et al. (2012) Developmental control of gene copy number by repression of replication initiation and fork progression. Genome Res 22:64-75

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