Jove
Visualize
Contact Us
JoVE
x logofacebook logolinkedin logoyoutube logo
ABOUT JoVE
OverviewLeadershipBlogJoVE Help Center
AUTHORS
Publishing ProcessEditorial BoardScope & PoliciesPeer ReviewFAQSubmit
LIBRARIANS
TestimonialsSubscriptionsAccessResourcesLibrary Advisory BoardFAQ
RESEARCH
JoVE JournalMethods CollectionsJoVE Encyclopedia of ExperimentsArchive
EDUCATION
JoVE CoreJoVE BusinessJoVE Science EducationJoVE Lab ManualFaculty Resource CenterFaculty Site
Terms & Conditions of Use
Privacy Policy
Policies

Related Experiment Videos

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
This summary is machine-generated.

Related Concept Videos

You might also read

Related Articles

Articles linked to this work by shared authors, journal, and citation graph.

Sort by
Same author

Werner Syndrome, aging and cancer.

Genome dynamics·2008
Same author

The lens eyes of the box jellyfish Tripedalia cystophora and Chiropsalmus sp. are slow and color-blind.

Journal of comparative physiology. A, Neuroethology, sensory, neural, and behavioral physiology·2007
Same author

Is the brain overrated?

The British journal of ophthalmology·2003
Same author

Mutations in human DNA polymerase eta motif II alter bypass of DNA lesions.

The EMBO journal·2001
Same author

In vivo mutagenesis by Escherichia coli DNA polymerase I. Ile(709) in motif A functions in base selection.

The Journal of biological chemistry·2001
Same author

Getting a grip on how DNA polymerases function.

Nature structural biology·2001

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.

Related Experiment Videos

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.