# EXCISION REPAIR OF UV IRRADIATED DNA IN YEAST

> **NIH NIH R01** · UNIVERSITY OF ROCHESTER · 1991 · $263,044

## Abstract

The long term goals are to elucidate the genetic and molecular
mechanism of excision repair of DNA damaged by ultraviolet (UV)
light irradiation in the yeast, Saccharomyces cerevisiae.  The
overall objective of this proposal is to examine the structure,
regulation, and function of the RAD1, RAD2, RAD3, RAD4, and RAD10
genes and their encoded proteins involved in the incision step of
excision repair, to reconstitute the incision activity in vitro,
and to define the mechanism of its action.  In addition, the
viability role of the RAD3 gene and protein will be determined.

The RAD4 gene is the only gene among these five genes that remains
to be cloned; this gene will be isolated from the yeast genome by
gap repair and its nucleotide sequence determined.  The regulation
of the RAD1, RAD2, RAD3, RAD4, and RAD10 genes and proteins will
be examined following treatment of yeast cells with DNA damaging
agents and during different cell cycle stages.  The cellular
location of the RAD1, RAD2, RAD3, RAD4, and RAD10 proteins will be
determined by indirect immunofluorescence.  The DNA dependent
ATPASE and helicase activities of the RAD3 protein will be further
characterized, and the role of RAD3 protein in recognition of the
damaged site in DNA examined.  To correlate the in vivo biological
functions with the biochemical activities, various RAD3 mutant
proteins will be overproduced and purified from yeast and
biochemically characterized.  The RAD1, RAD2, RAD4, and RAD10
proteins will be overproduced and purified from yeast cells and
examined for DNA binding, ATPase, DNA unwindling, endonuclease and
exonuclease activities.  Combinations of RAD proteins will be
examined for various biochemical activities and for incision of UV
damaged DNA, and the mechanism of incision determined.  To
understand the essential role of the RAD3 gene in cell viability,
heat sensitive (ts) mutations will be isolated and examined for
effects on DNA replication, cell cycle, recombination, and other
cellular processes.  Genes coding for proteins that interact with
the RAD3 protein in its viability function will be identified by
isolating extragenic allele-specific cold sensitive (cs)
suppressors of RAD3 ts mutations, and the biological roles of these
cs suppressor genes determined.  The wild type allele of cs
suppressor genes will be cloned by complementation of the cs
mutation and the structure, regulation, and function of these
suppressor genes studied.

Cells from xeroderma pigmentosum patients are defective in excision
repair of UV induced pyrimidine dimers from DNA and these patients
are prone to develop skin cancers, indicating a relationship
between defective DNA repair and carcinogenesis.  The proposed
studies should provide a model system for understanding the
mechanism of excision repair in eukaryotes, including in humans.

## Key facts

- **NIH application ID:** 3172790
- **Project number:** 5R01CA035035-09
- **Recipient organization:** UNIVERSITY OF ROCHESTER
- **SAM.gov UEI:** F27KDXZMF9Y8
- **Principal Investigator:** SATYA PRAKASH
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 1991
- **Award amount:** $263,044
- **Award type:** 5
- **Project period:** 1983-07-01 → 1993-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/3172790

## Citation

> US National Institutes of Health, RePORTER application 3172790, EXCISION REPAIR OF UV IRRADIATED DNA IN YEAST (5R01CA035035-09). Retrieved via MORVS 2026-10-25 from https://api.morvs.ai/grant/nih/3172790. Licensed CC0.

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