# OPERATION OF A DNA REPAIR PATHWAY IN VITRO

> **NIH NIH R01** · UNIV OF NORTH CAROLINA CHAPEL HILL · 1988 · $91,869

## 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.

## Key facts

- **NIH application ID:** 3174542
- **Project number:** 2R01CA036906-04
- **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:** 1988
- **Award amount:** $91,869
- **Award type:** 2
- **Project period:** 1985-09-01 → 1990-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/3174542

## Citation

> US National Institutes of Health, RePORTER application 3174542, OPERATION OF A DNA REPAIR PATHWAY IN VITRO (2R01CA036906-04). Retrieved via MORVS 2026-10-25 from https://api.morvs.ai/grant/nih/3174542. Licensed CC0.

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