Abstract

Poly(ADP-ribose) (pADPR) is present in the nuclei of all eukaryotic organisms. Although a number of studies have suggested an involvement of this polymer in repair of damage produced by ionizing radiation and other agents, the biological role of the polymer remains unclear. Thus, the ultimate goal of this research is to determine what the nature of this involvement in repair might be in mammalian cells. To answer this question we have isolated Chinese hamster ovary cell mutants (PADR) that are deficient in pADPR synthesis and find that these mutants are also abnormally sensitive to DNA alkylating agents and possibly to gamma rays. To determine the basis of this sensitivity, PADR mutant cells will be examined for increased frequency of DNA damage, defects in excision repair, single and double strand DNA breaks, and repair replication. An increase in frequency in sister-chromatid exchanges in PADR mutants will also be examined as will as a reduction in NAD and ATP pools. The possibility of an alteration in the mutant's chromatin structure will be examined by determining the pattern of EMS-induced histone poly ADP-ribosylation using 2 dimensional gel electrophoresis. The mutant's nuclear matrix distribution of poly (ADP-ribose) will be examined as well as an alteration in the mutant's sensitivity to electroporated restriction enzyme. Mutants deficient in poly(ADP-ribose) glycohydolase will be isolated by screening for colonies that remain 32P-labeled after inhibition of poly(ADP-ribose) synthesis with 3-aminobenzamide. Mutants defective in the induction of poly(ADP-ribose) synthesis by DNA breaks will be isolated by screening colonies for poly(ADP-ribose) polymerase activity after gamma-irradiation and isolating colonies that do not synthesize the polymer. Poly(ADP-ribose) polymerase activity in colonies is detected using labeling with 32P-NAD and autoradiography. We believe that biochemical characterization of poly(ADP-ribose) deficient mutants offers the prospect of elucidating the role of this polymer in DNA repair.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
2R01CA048636-05
Application #
3192526
Study Section
Radiation Study Section (RAD)
Project Start
1988-05-01
Project End
1994-07-31
Budget Start
1991-08-01
Budget End
1992-07-31
Support Year
5
Fiscal Year
1991
Total Cost
Indirect Cost

  Institution

Name
Lankenau Institute for Medical Research
Department
Type
DUNS #
125797084
City
Wynnewood
State
PA
Country
United States
Zip Code
19096