The reaction mechanism of DNA glycosylase/AP lyases at abasic sites

Amanda McCullough, A. Sanchez, M. L. Dodson, P. Marapaka, J. S. Taylor, Robert (Stephen) Lloyd

Research output: Contribution to journalArticle

42 Citations (Scopus)

Abstract

DNA glycosylase and glycosylase/abasic (AP) lyases are the enzymes responsible for initiating the base excision repair pathway by recognizing the damaged target base and catalyzing the breakage of the base - sugar glycosyl bond. The subset of glycosylases that have an associated AP lyase activity also catalyze DNA strand breakage at the resulting or preexisting AP site via a β-elimination reaction, proceeding from an enzyme - DNA imino intermediate. Two distinct mechanisms have been proposed for the formation of this intermediate. These mechanisms essentially differ in the nature of the first bond broken and the timing of the opening of the deoxyribose ring. The data presented here demonstrate that the combined rate of sugar ring opening and reduction of the sugar is significantly slower than the rate of formation of a T4-pyrimidine dimer glycosylase (T4-pdg) - DNA intermediate. Using a methyl-deoxyribofuranose AP-site analogue that is incapable of undergoing sugar ring opening, it was demonstrated that the T4-pdg reaction can initiate at the ring-closed form, albeit at a drastically reduced rate. T4-pdg preferentially cleaved the β-anomer of the methyldeoxyribofuranose AP site analogue. This is consistent with a mechanism in which the methoxy group is backside-displaced by the amino group from the α-face of the deoxyribofuranose ring. In addition, studies examining rates of sugar-aldehyde reduction and the sodium borohydride concentration dependence of the rate of formation of the covalent imine intermediate suggest that the reduction of the intermediate is rate-limiting in the reaction.

Original languageEnglish (US)
Pages (from-to)561-568
Number of pages8
JournalBiochemistry
Volume40
Issue number2
DOIs
StatePublished - Jan 16 2001
Externally publishedYes

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DNA-(Apurinic or Apyrimidinic Site) Lyase
DNA Glycosylases
Pyrimidine Dimers
Sugars
Deoxyribose
Imines
DNA
Enzymes
Aldehydes
DNA Repair
Repair
sodium borohydride
deoxyribopyrimidine endonucleosidase

ASJC Scopus subject areas

  • Biochemistry

Cite this

The reaction mechanism of DNA glycosylase/AP lyases at abasic sites. / McCullough, Amanda; Sanchez, A.; Dodson, M. L.; Marapaka, P.; Taylor, J. S.; Lloyd, Robert (Stephen).

In: Biochemistry, Vol. 40, No. 2, 16.01.2001, p. 561-568.

Research output: Contribution to journalArticle

McCullough, Amanda ; Sanchez, A. ; Dodson, M. L. ; Marapaka, P. ; Taylor, J. S. ; Lloyd, Robert (Stephen). / The reaction mechanism of DNA glycosylase/AP lyases at abasic sites. In: Biochemistry. 2001 ; Vol. 40, No. 2. pp. 561-568.
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