# OPERATION OF A DNA EXCISION REPAIR PATHWAY IN VITRO

> **NIH NIH R01** · UNIV OF NORTH CAROLINA CHAPEL HILL · 1987 · $83,492

## Abstract

The objectives of the project are to describe and define the operations of
a DNA excision repair pathway in carcinogen-damaged human fibroblasts
rendered permeable to enzymes, substrates and cofactors.  Cells will be
treated with 254 nm radiation or with
(+-)-7alpha,8beta-dihydroxy-9beta,10beta-epoxy-7,8,9,10-tetrahydrobenzo(a)py
rene to damage DNA, then permeabilized and incubated in vitro for assay of
each step of excision repair.  The conditions for assaying the strand
incision step of repair in permeable cells will be optimized with attention
directed to maximizing the signal of damage-directed strand incisions while
minimizing non-specific DNA breakage.  Reparative strand incisions, release
of damaged DNA, gap-filling DNA synthesis and strand ligation all will be
quantified in permeable cells and the efficiency of operation of each step
compared with what is seen in intact cells.  The sensitivity to digestion
by micrococcal nuclease of repair patches synthesized in permeable cells
will also be determined.  The DNA repair assays will then be performed
using permeable xeroderma pigmentosum cells with moderate or severe
reparative deficiencies in order to quantify the reparative deficit in
vitro.  DNA-free human cell extracts or purified prokaryotic endonucleases
will be added to deficient permeable cells to try to restore or stimulate
reparative activity.  Successful operation of repair in vitro will
facilitate the characterization of enzymatic activities that are required
of nucleotidyl DNA excision repair in human cells.

## Key facts

- **NIH application ID:** 3174546
- **Project number:** 5R01CA036906-03
- **Recipient organization:** UNIV OF NORTH CAROLINA CHAPEL HILL
- **SAM.gov UEI:** D3LHU66KBLD5
- **Principal Investigator:** William K. Kaufmann
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 1987
- **Award amount:** $83,492
- **Award type:** 5
- **Project period:** 1985-09-01 → 1988-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/3174546

## Citation

> US National Institutes of Health, RePORTER application 3174546, OPERATION OF A DNA EXCISION REPAIR PATHWAY IN VITRO (5R01CA036906-03). Retrieved via MORVS 2026-10-25 from https://api.morvs.ai/grant/nih/3174546. Licensed CC0.

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