Analysis of nucleotide excision repair by detection of single-stranded DNA transients.

Abstract:

:Nucleotide excision repair (NER) removes bulky DNA lesions and is thus crucial for the protection against environmental carcinogens and UV light exposure. Deficiencies in NER cause increased mutation rates and chromosomal aberrations. Current methods for studying NER are mostly based on either quantitation of lesion removal or detection of repair DNA synthesis. Both have their limitations: lesion removal is inaccurate at very short times post-lesion, where the fraction of removal is low. Repair synthesis is difficult to apply to normally cycling cells due to the need to discriminate repair from replicative DNA synthesis. To overcome these problems we developed a method for analysis of NER based on detection of transient single-stranded (ss) DNA stretches generated at the nucleotide excision step. Cells are metabolically labelled with BrdU, exposed to UV-irradiation and the ssDNA transients generated during excision repair are detected using an anti-BrdU antibody. The method allows single-cell microscopic analysis of the distribution of DNA repair sites as well as kinetic analysis of the DNA repair response. Studies using various DNA repair-deficient cell lines indicate that the detection method integrates a number of pre-synthesis nucleotide excision repair stages. Thus, assembled repair sites can be detected even when they may not lead to complete resolution of the DNA lesion. Using this approach, we show that repair helicase-deficient cells differ from endonuclease-deficient cells.

journal_name

Carcinogenesis

journal_title

Carcinogenesis

authors

Rubbi CP,Milner J

doi

10.1093/carcin/22.11.1789

keywords:

subject

Has Abstract

pub_date

2001-11-01 00:00:00

pages

1789-96

issue

11

eissn

0143-3334

issn

1460-2180

journal_volume

22

pub_type

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