6-Carboxyfluorescein and structurally similar molecules inhibit DNA binding and repair by O 6-alkylguanine DNA alkyltransferase

Manana Melikishvili, David W. Rodgers, Michael G. Fried

Research output: Contribution to journalArticlepeer-review

15 Scopus citations

Abstract

Human O 6-alkylguanine-DNA alkyltransferase (AGT) repairs mutagenic O 6-alkylguanine and O 4-alkylthymine adducts in single-stranded and duplex DNAs. These activities protect normal cells and tumor cells against drugs that alkylate DNA; drugs that inactivate AGT are under test as chemotherapeutic enhancers. In studies using 6-carboxyfluorescein (FAM)-labeled DNAs, AGT reduced the fluorescence intensity by ~40% at binding saturation, whether the FAM was located at the 5′ or the 3′ end of the DNA. AGT protected residual fluorescence from quenching, indicating a solute-inaccessible binding site for FAM. Sedimentation equilibrium analyses showed that saturating AGT-stoichiometries were higher with FAM-labeled DNAs than with unlabeled DNAs, suggesting that the FAM provides a protein binding site that is not present in unlabeled DNAs. Additional fluorescence and sedimentation measurements showed that AGT forms a 1:1 complex with free FAM. Active site benzylation experiments and docking calculations support models in which the primary binding site is located in or near the active site of the enzyme. Electrophoretic analyses show that FAM inhibits DNA binding (IC 50~76μM) and repair of DNA containing an O 6-methylguanine residue (IC 50~63μM). Similar results were obtained with other polycyclic aromatic compounds. These observations demonstrate the existence of a new class of non-covalent AGT-inhibitors. After optimization for binding-affinity, members of this class might be useful in cancer chemotherapy.

Original languageEnglish
Pages (from-to)1193-1202
Number of pages10
JournalDNA Repair
Volume10
Issue number12
DOIs
StatePublished - Dec 10 2011

Bibliographical note

Funding Information:
We thank the reviewers for their thoughtful suggestions. This work was supported by NIH grants NS-38041 and P20 RR-20171 to D.W.R. and GM-070662 to M.G.F.

Keywords

  • Analytical ultracentrifugation
  • DNA alkyltransferase
  • DNA repair
  • EMSA
  • Fluorescein
  • Fluorescence assay
  • Substrate analogue

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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