Measurement of oxidative DNA damage and repair in specific DNA sequences.

We describe two methods that were developed in our laboratory to measure the production and repair of oxidative DNA damage in specific DNA sequences. Both of these methods rely on the use of monoclonal antibodies against modified nucleotides to separate DNA sequences that contain damage from those in which repair has occurred. In one case, the modified base is bromodeoxyuridine, which is inserted into the DNA during the repair synthesis step of excision repair.

Transcription-coupled repair of 8-oxoguanine: requirement for XPG, TFIIH, and CSB and implications for Cockayne syndrome.

Analysis of transcription-coupled repair (TCR) of oxidative lesions here reveals strand-specific removal of 8-oxo-guanine (8-oxoG) and thymine glycol both in normal human cells and xeroderma pigmentosum (XP) cells defective in nucleotide excision repair. In contrast, Cockayne syndrome (CS) cells including CS-B, XP-B/CS, XP-D/CS, and XP-G/CS not only lack TCR but cannot remove 8-oxoG in a transcribed sequence, despite its proficient repair when not transcribed. The XP-G/CS defect uniquely slows lesion removal in nontranscribed sequences.

Growth retardation, DNA repair defects, and lack of spermatogenesis in BRCA1-deficient mice.

BRCA1 is a nuclear phosphoprotein expressed in a broad spectrum of tissues during cell division. The inheritance of a mutant BRCA1 allele dramatically increases a woman's lifetime risk for developing both breast and ovarian cancers. A number of mouse lines carrying mutations in the Brca1 gene have been generated, and mice homozygous for these mutations generally die before day 10 of embryonic development.

BRCA1 required for transcription-coupled repair of oxidative DNA damage.

The breast and ovarian cancer susceptibility gene BRCA1 encodes a zinc finger protein of unknown function. Association of the BRCA1 protein with the DNA repair protein Rad51 and changes in the phosphorylation and cellular localization of the protein after exposure to DNA-damaging agents are consistent with a role for BRCA1 in DNA repair. Here, it is shown that mouse embryonic stem cells deficient in BRCA1 are defective in the ability to carry out transcription-coupled repair of oxidative DNA damage, and are hypersensitive to ionizing radiation and hydrogen peroxide.

Requirement for DNA mismatch repair proteins in the transcription-coupled repair of thymine glycols in Saccharomyces cerevisiae.

Defects in DNA mismatch repair have been shown to lead to increased genomic instability and mutability. We recently found that human cells defective in the DNA mismatch repair gene, hMSH2, were deficient in the transcription-coupled repair (TCR) of both oxidative DNA damage, including thymine glycols, and UV-induced DNA damage. However, in a hMLH1 mutant, only a reduction in the TCR of UV damage was observed.

Differential involvement of the human mismatch repair proteins, hMLH1 and hMSH2, in transcription-coupled repair.

Defects in DNA mismatch repair have been associated with both hereditary and sporadic forms of cancer. Recently, it has been shown that human cell lines deficient in mismatch repair were also defective in the transcription-coupled repair (TCR) of UV-induced DNA damage. We examined whether TCR of ionizing radiation-induced DNA damage also requires the genes involved in DNA mismatch repair.

Evaluation of adjuvants that enhance the effectiveness of antisense oligodeoxynucleotides.

Purpose: A factor limiting the effectiveness of antisense (AS) deoxyoligonucleotides (ODNs) is inefficient transport to their sites of action in the cytoplasm and in the nucleus. The extent of ODN transfer from endosomes to cytosol seems to be an important determinant of ODN effects. Consequently, the development of compounds (adjuvants) that enhance endosome to cytosol transfer may be vital in AS ODN therapeutics.

Radiolabeling of methylphosphonate and phosphorothioate oligonucleotides and evaluation of their transport in everted rat jejunum sacs.

Purpose: The therapeutic use of antisense oligonucleotides will likely involve their administration over protracted periods of time. The oral route of drug dosing offers many advantages over other possible routes when chronic drug administration is necessary. However, little is known about the potential for oligonucleotide uptake from the gastrointestinal tract.

Influence of base composition on membrane binding and cellular uptake of 10-mer phosphorothioate oligonucleotides in Chinese hamster ovary (CHRC5) cells.

A key problem in antisense therapeutics is the relatively poor cell uptake of oligonucleotides and subsequent transport to the cytoplasm and nucleus. Although the chemical characteristics of oligonucleotides seem likely to affect their uptake by cells, little is known about this issue. In this article we explore the effect of base composition on oligonucleotide uptake.