Repair of O6-G-alkyl-O6-G interstrand cross-links by human O6-alkylguanine-DNA alkyltransferase

Qingming Fang, Anne M. Noronha, Sebastian P. Murphy, Christopher J. Wilds, Julie L. Tubbs, John A. Tainer, Goutam Chowdhury, F. Peter Guengerich, Anthony E. Pegg

Research output: Contribution to journalArticle

34 Citations (Scopus)

Abstract

O6-Alkylguanine-DNA alkyltransferase (AGT) plays an important role by protecting cells from alkylating agents. This reduces the frequency of carcinogenesis and mutagenesis initiated by such agents, but AGT also provides a major resistance mechanism to some chemotherapeutic drugs. To improve our understanding of the AGT-mediated repair reaction and our understanding of the spectrum of repairable damage, we have studied the ability of AGT to repair interstrand cross-link DNA damage where the two DNA strands are joined via the guanine-O6 in each strand. An oligodeoxyribonucleotide containing a heptane cross-link was repaired with initial formation of an AGT-oligo complex and further reaction of a second AGT molecule yielding a hAGT dimer and free oligo. However, an oligodeoxyribonucleotide with a butane cross-link was a very poor substrate for AGT-mediated repair, and only the first reaction that forms an AGT-oligo complex could be detected. Models of the reaction of these substrates in the AGT active site show that the DNA duplex is forced apart locally to repair the first guanine. This reaction is greatly hindered with the butane cross-link, which is mostly buried in the active site pocket and limited in conformational flexibility. This limitation also prevents the adoption of a conformation for the second reaction to repair the AGT-oligo complex. These results are consistent with the postulated mechanism of AGT repair that involves DNA binding and flipping of the substrate nucleotide and indicate that hAGT can repair some types of interstrand cross-link damage.

Original languageEnglish (US)
Pages (from-to)10892-10903
Number of pages12
JournalBiochemistry
Volume47
Issue number41
DOIs
StatePublished - Oct 14 2008

Fingerprint

Oligodeoxyribonucleotides
Guanine
Catalytic Domain
Repair
Heptanes
Alkylating Agents
DNA
Mutagenesis
DNA Repair
DNA Damage
Carcinogenesis
Nucleotides
Pharmaceutical Preparations
Substrates
butane
DNA alkyltransferase
Dimers
Conformations
Molecules

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Fang, Q., Noronha, A. M., Murphy, S. P., Wilds, C. J., Tubbs, J. L., Tainer, J. A., ... Pegg, A. E. (2008). Repair of O6-G-alkyl-O6-G interstrand cross-links by human O6-alkylguanine-DNA alkyltransferase. Biochemistry, 47(41), 10892-10903. https://doi.org/10.1021/bi8008664
Fang, Qingming ; Noronha, Anne M. ; Murphy, Sebastian P. ; Wilds, Christopher J. ; Tubbs, Julie L. ; Tainer, John A. ; Chowdhury, Goutam ; Guengerich, F. Peter ; Pegg, Anthony E. / Repair of O6-G-alkyl-O6-G interstrand cross-links by human O6-alkylguanine-DNA alkyltransferase. In: Biochemistry. 2008 ; Vol. 47, No. 41. pp. 10892-10903.
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Fang, Q, Noronha, AM, Murphy, SP, Wilds, CJ, Tubbs, JL, Tainer, JA, Chowdhury, G, Guengerich, FP & Pegg, AE 2008, 'Repair of O6-G-alkyl-O6-G interstrand cross-links by human O6-alkylguanine-DNA alkyltransferase', Biochemistry, vol. 47, no. 41, pp. 10892-10903. https://doi.org/10.1021/bi8008664

Repair of O6-G-alkyl-O6-G interstrand cross-links by human O6-alkylguanine-DNA alkyltransferase. / Fang, Qingming; Noronha, Anne M.; Murphy, Sebastian P.; Wilds, Christopher J.; Tubbs, Julie L.; Tainer, John A.; Chowdhury, Goutam; Guengerich, F. Peter; Pegg, Anthony E.

In: Biochemistry, Vol. 47, No. 41, 14.10.2008, p. 10892-10903.

Research output: Contribution to journalArticle

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AU - Fang, Qingming

AU - Noronha, Anne M.

AU - Murphy, Sebastian P.

AU - Wilds, Christopher J.

AU - Tubbs, Julie L.

AU - Tainer, John A.

AU - Chowdhury, Goutam

AU - Guengerich, F. Peter

AU - Pegg, Anthony E.

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N2 - O6-Alkylguanine-DNA alkyltransferase (AGT) plays an important role by protecting cells from alkylating agents. This reduces the frequency of carcinogenesis and mutagenesis initiated by such agents, but AGT also provides a major resistance mechanism to some chemotherapeutic drugs. To improve our understanding of the AGT-mediated repair reaction and our understanding of the spectrum of repairable damage, we have studied the ability of AGT to repair interstrand cross-link DNA damage where the two DNA strands are joined via the guanine-O6 in each strand. An oligodeoxyribonucleotide containing a heptane cross-link was repaired with initial formation of an AGT-oligo complex and further reaction of a second AGT molecule yielding a hAGT dimer and free oligo. However, an oligodeoxyribonucleotide with a butane cross-link was a very poor substrate for AGT-mediated repair, and only the first reaction that forms an AGT-oligo complex could be detected. Models of the reaction of these substrates in the AGT active site show that the DNA duplex is forced apart locally to repair the first guanine. This reaction is greatly hindered with the butane cross-link, which is mostly buried in the active site pocket and limited in conformational flexibility. This limitation also prevents the adoption of a conformation for the second reaction to repair the AGT-oligo complex. These results are consistent with the postulated mechanism of AGT repair that involves DNA binding and flipping of the substrate nucleotide and indicate that hAGT can repair some types of interstrand cross-link damage.

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Fang Q, Noronha AM, Murphy SP, Wilds CJ, Tubbs JL, Tainer JA et al. Repair of O6-G-alkyl-O6-G interstrand cross-links by human O6-alkylguanine-DNA alkyltransferase. Biochemistry. 2008 Oct 14;47(41):10892-10903. https://doi.org/10.1021/bi8008664