Abstract

This is the continuation of a project to study the operation of the nucleotidyl DNA excision repair pathway (NDERP) in vitro. In the first two years of the project we have characterized UV- dependent and non-specific endodeoxyribonuclease activities in permeable human fibroblasts using alkaline elution methodology to quantify DNA strand breaks. An ATP-dependent and UV- dependent endodeoxyribonuclease activity was observed in normal and xeroderma pigmentosum (XP) variant fibroblasts which represents the rate-limiting, dimer-directed incision step in the NDERP. This activity was undetectable in XP cells from complementation group A. A second UV-endodeoxyribonuclease activity which did not require ATP was identified in all human fibroblast strains which represents the redoxy endodeoxyribonuclease that cuts DNA at ring-saturated pyrimidines. Optimal conditions for assay of the UV- endodeoxyribonucleases were established. Various preparations of cellular extracts were added to permeable XPA to test for in vitro complementation of the defect in dimer-directed incision. UV-dependent incision could not be consistently demonstrated and non-specific endodeoxyribonuclease activities contaminated most cell extracts. Consequently, it appears that restoration of repair in permeable XPA will require fractionation of extracts to remove non-specific endodeoxyribonuclease. This fractionation will be facilitated by establishment of a plasmid-based endodeoxyribonuclease assay that can distinguish non-specific endodeoxyribonucleases from the two specific UV- endodeoxyribonucleases. A plasmid incision assay will be used to identify and isolate the dimer-directed UV-endodeoxyribonuclease from repair-proficient human cells. We will then determine whether incision-defective XPA also express this endodeoxyribonuclease. The isolated enzyme will be added to permeable XPA to test for restoration of dimer-directed incision of nuclear DNA. Peculiarities of reparative incision as demonstrated by other repair-deficient complementation groups of XP also will be examined. These studies will aid in identifying the bases of the various reparative defects seen in XP.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
2R01CA036906-04
Application #
3174542
Study Section
Chemical Pathology Study Section (CPA)
Project Start
1985-09-01
Project End
1990-08-31
Budget Start
1988-09-01
Budget End
1989-08-31
Support Year
4
Fiscal Year
1988
Total Cost
Indirect Cost

  Institution

Name
University of North Carolina Chapel Hill
Department
Type
Schools of Medicine
DUNS #
078861598
City
Chapel Hill
State
NC
Country
United States
Zip Code
27599