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Engineering human DNA alkyltransferases for gene therapy using random sequence mutagenesis

L P Encell1, M M Coates, L A Loeb

  • 1Department of Pathology, University of Washington School of Medicine, Seattle 98195-7705, USA.

Cancer Research
|March 21, 1998
PubMed
Summary
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Researchers engineered O6-Alkylguanine-DNA alkyltransferase (AGT) mutants for enhanced resistance to DNA alkylation damage. These novel AGT variants show promise for gene therapy, protecting healthy cells during cancer treatment.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Genetics

Background:

  • O6-Alkylguanine-DNA alkyltransferase (AGT) is a crucial DNA repair enzyme.
  • Understanding AGT's active site is key to developing cancer therapies.
  • Current therapies use alkylating agents, which AGT counteracts.

Purpose of the Study:

  • To generate and characterize human AGT mutants with resistance to both alkylating agents and AGT inhibitors.
  • To explore the potential of these mutants in gene therapy applications.

Main Methods:

  • Random mutagenesis was used to create a large library of human AGT mutants.
  • Mutants were selected for resistance to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and O6-benzylguanine (BG) in E. coli.
  • Surviving clones were sequenced to identify mutations.

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Main Results:

  • Multiple amino acid substitutions, often at nonconserved positions, conferred resistance.
  • Nine selected mutants exhibited up to 341-fold higher survival than wild-type AGT under MNNG/BG treatment.
  • Increased substitutions correlated with enhanced resistance to MNNG and BG.

Conclusions:

  • Extensive mutagenesis can yield highly BG-resistant AGT variants.
  • These BG-resistant AGT mutants are promising candidates for gene therapy to protect normal tissues during cancer treatment with alkylating agents and BG.