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A Simple, Rapid, and Quantitative Assay to Measure Repair of DNA-protein Crosslinks on Plasmids Transfected into Mammalian Cells
11:58

A Simple, Rapid, and Quantitative Assay to Measure Repair of DNA-protein Crosslinks on Plasmids Transfected into Mammalian Cells

Published on: March 5, 2018

Evidence for a recombination-independent pathway for the repair of DNA interstrand cross-links based on a

M Berardini1, W Mackay, E L Loechler

  • 1Department of Biology, Boston University, Massachusetts 02215, USA.

Biochemistry
|March 25, 1997
PubMed

Insights

DNA-DNA interstrand cross-links are repaired independently of recombination. Nucleotide excision repair (NER) is involved, but other common repair pathways are not, suggesting a novel repair mechanism for these DNA cross-links.

Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • DNA-DNA interstrand cross-links (ICLs) are critical cytotoxic lesions induced by various chemotherapeutic agents.
  • Understanding the repair mechanisms of ICLs is crucial for developing effective cancer therapies.
  • Previous models proposed that ICL repair involves both nucleotide excision repair (NER) and recombination pathways.

Purpose of the Study:

  • To investigate the repair pathways of nitrogen mustard-induced DNA interstrand cross-links (ICLs).
  • To determine the roles of recombination and other DNA repair pathways in ICL tolerance.
  • To elucidate the mechanism of replication efficiency following ICL induction.

Main Methods:

  • Site-specific construction of a plasmid containing a single nitrogen mustard ICL (inter-HN2-pTZSV28).
  • Transformation of the modified plasmid into various strains of Escherichia coli, including those deficient in specific repair pathways (e.g., recA, NER enzymes, base excision repair enzymes, 5'→3' exonucleases).
  • Measurement of replication efficiency (RE) by determining colony-forming ability.

Main Results:

  • A replication efficiency of approximately 0.3 was observed in wild-type E. coli, indicating ICL repair.
  • ICL repair was independent of the RecA protein and other recombination pathways (RE ≈ 0.3 in delta recA strain).
  • Nucleotide excision repair (NER) was found to be essential for ICL repair, as RE decreased to approximately 0.02 in NER-deficient strains.
  • Base excision repair and 5'→3' exonuclease activities were not found to be significantly involved in ICL repair.
  • Psoralen ICLs were not repaired by the identified NER-dependent pathway.

Conclusions:

  • DNA interstrand cross-links induced by nitrogen mustard are repaired via a pathway that is independent of recombination.
  • Nucleotide excision repair (NER) plays a crucial role in this recombination-independent ICL repair.
  • The findings suggest a novel DNA repair pathway for ICLs, potentially involving NER followed by replicative bypass, with implications for understanding the differential cytotoxicity of various ICL-inducing agents.

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