Sequence-specific and Domain-specific DNA Repair in Xeroderma Pigmentosum and Cockayne Syndrome Cells

Yuqing Tu,Steven Bates,Gerd P Pfeifer

doi:10.1074/jbc.272.33.20747

Yuqing Tu, Steven Bates + Show 1 more

Open Access

https://doi.org/10.1074/jbc.272.33.20747

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Abstract

Xeroderma pigmentosum (XP) and Cockayne syndrome (CS) cells have specific DNA repair defects. We had previously analyzed repair rates of cyclobutane pyrimidine dimers at nucleotide resolution along the human JUN gene in normal fibroblasts and found very efficient repair of sequences near the transcription initiation site but slow repair along the promoter. To investigate sequence-specific repair rate patterns in XP and CS cells, we conducted a similar analysis in XPA, XPB, XPC, XPD, and CSB fibroblasts. XPA cells were almost completely repair-deficient at all sequences analyzed. XPC cells repaired only the transcribed DNA strand beginning at position -20 relative to the transcription start site. Both XBP and XPD cells were deficient in repair of nontranscribed DNA and also very inefficiently repaired the transcribed strand including sequences near the transcription start site. CSB cells exhibited rapid repair near the transcription initiation site but were deficient in repair of sequences encountered by RNA polymerase during elongation (beginning at position +20). Since transcription of the JUN gene was UV-induced in all fibroblast strains, including CSB, the defective repair of the transcribed strand in CSB cannot be explained by a lack of transcription; rather, it appears to be a true DNA repair defect.

Highlights

Ultraviolet irradiation induces two major types of photoproducts in DNA, the cyclobutane pyrimidine dimers (CPDs)1 and the pyrimidine (6 – 4)-pyrimidone photoproducts, as well as much lower amounts of purine dimers, pyrimidine monoadducts, and a photoproduct between adjacent A and T bases
Since transcription of the JUN gene was UV-induced in all fibroblast strains, including CSB, the defective repair of the transcribed strand in CSB cannot be explained by a lack of transcription; rather, it appears to be a true DNA repair defect
Quantitation of Repair Rates—Nylon membranes were exposed to a PhosphorImager (Molecular Dynamics, Sunnyvale, CA), and radioactivity was determined in all CPD-specific bands of the sequencing gel that showed a consistent and measurable signal above background

Summary

EXPERIMENTAL PROCEDURES

Cell Culture and UV Irradiation—The following repair-deficient human fibroblast strains were obtained from the NIGMS (National Institutes of Health) human genetic mutant cell repository: GM00710B (XPA), GM13025 (XPB), GM00676 (XPC), GM10428 (XPD), and GM01098B (CSB). For DNA repair experiments, the original medium was returned to the cells, and the cells were incubated for various periods of time before lysis and DNA analysis. The oligonucleotide primer set JC, which is specific for sequences of the human JUN gene, was used to map CPDs in the JUN promoter along sequences surrounding the transcription initiation site, and along the transcribed DNA strand [15]. Quantitation of Repair Rates—Nylon membranes were exposed to a PhosphorImager (Molecular Dynamics, Sunnyvale, CA), and radioactivity was determined in all CPD-specific bands of the sequencing gel that showed a consistent and measurable signal above background. The time at which 50% of the initial damage was removed was determined from this curve These values were incorporated into Fig. 9

RESULTS

Findings

DISCUSSION