Patients with tuberous sclerosis complex and autosomal dominant polycystic kidney disease most often are born with anatomically normal kidneys but develop significant renal involvement as they age. The abnormal tissues in these diseases are associated with the loss of heterozygosity (LOH) such that only the defective allele is present at the disease locus. Although both diseases have a second associated gene, the TSC2 and PKD1 genes cause a more severe phenotype and are more often found in patients with new mutations. We postulate that the disease severity is related to the fact that these adjacent genes are in an unstable region of chromosome 16. We present evidence that inverted Alu repeats and polypurine.polypyrimidine tracts from these genes appear to be associated with deletions, and block the human replication fork. In addition, the polypurine.polypyrimidine tract, under conditions that favor alternative secondary structure formation, can spontaneously initiate replication. Such replication initiation and termination phenomenon have both developmental and mutagenic implications. The ultimate goal of our reseach is to retard disease onset and progression by delaying the second somatic mutation leading to the LOH in the TSC2 and PKD1 genes. We hypothesize that alternative DNA secondary structures in the TSC2 and PKD1 genes promote mutagenesis through their effects on DNA replication. The proposed studies investigate the DNA structural characteristics of the Pu.Py tracts and inverted Alu repeats using 2-dimensional gel and melting curve analyses. Using repair deficient cell lines, we will also determine the replication proteins involved. Using a stable-transfection system, we will measure the ability of the sequences to stall replication and to induce recombination. The potential to initiate the human replication fork will also be studied in a well characterized system. By understanding the effects of these sequences on the fidelity of DNA replication, therapeutic inroads into delaying disease onset can be made.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
1R01DK061458-01A1
Application #
6576325
Study Section
General Medicine B Study Section (GMB)
Program Officer
Rasooly, Rebekah S
Project Start
2003-05-01
Project End
2008-02-28
Budget Start
2003-05-01
Budget End
2004-02-29
Support Year
1
Fiscal Year
2003
Total Cost
$306,342
Indirect Cost
Name
Cincinnati Children's Hospital Medical Center
Department
Type
DUNS #
071284913
City
Cincinnati
State
OH
Country
United States
Zip Code
45229
Bissler, John J (2015) Therapies for polycystic kidney disease. Curr Opin Pediatr 27:227-32
Bell, P Darwin; Fitzgibbon, Wayne; Sas, Kelli et al. (2011) Loss of primary cilia upregulates renal hypertrophic signaling and promotes cystogenesis. J Am Soc Nephrol 22:839-48
Dixon, Bradley P; Hulbert, John C; Bissler, John J (2011) Tuberous sclerosis complex renal disease. Nephron Exp Nephrol 118:e15-20
Kavanaugh, Gina M; Wise-Draper, Trisha M; Morreale, Richard J et al. (2011) The human DEK oncogene regulates DNA damage response signaling and repair. Nucleic Acids Res 39:7465-76
Dixon, Bradley P; Henry, Jeff; Siroky, Brian J et al. (2011) Cell cycle control and DNA damage response of conditionally immortalized urothelial cells. PLoS One 6:e16595
Liu, Guoqi; Chen, Xiaomi; Bissler, John J et al. (2010) Replication-dependent instability at (CTG) x (CAG) repeat hairpins in human cells. Nat Chem Biol 6:652-9
Siroky, Brian J; Czyzyk-Krzeska, Maria F; Bissler, John J (2009) Renal involvement in tuberous sclerosis complex and von Hippel-Lindau disease: shared disease mechanisms? Nat Clin Pract Nephrol 5:143-56
Dixon, Bradley P; Chu, Albert; Henry, Jeff et al. (2009) Increased cancer risk of augmentation cystoplasty: possible role for hyperosmolal microenvironment on DNA damage recognition. Mutat Res 670:88-95
Dixon, Bradley P; Lu, Lu; Chu, Albert et al. (2008) RecQ and RecG helicases have distinct roles in maintaining the stability of polypurine.polypyrimidine sequences. Mutat Res 643:20-8
Robison, Jacob G; Bissler, John J; Dixon, Kathleen (2007) Replication protein A is required for etoposide-induced assembly of MRE11/RAD50/NBS1 complex repair foci. Cell Cycle 6:2408-16

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