MECHANISM OF 06 -ALKYLGUANINE -DNA ALKYLTRANSFERASE

Project: Research project

Project Details

Description

O6-Alkylguanine-DNA alkyltransferase repairs damaged DNA by transferring an
alkyl chain from the O6-position of guanine to a cysteine on the protein.
This action produces the original DNA but the enzyme's free cysteine is not
regenerated and the enzyme activity is destroyed. The formation and
persistence of the O6-methylguanine lesion has been implicated in
carcinogenesis. The mechanism by which the protein effects methyl transfer
is not known.

The postulated mechanism of action of AGT which will be tested in this
proposal is as follows. AGT activates the cysteine as a nucleophile by
deprotonation. The guanine is enhanced as a leaving group by protonation
of a heteroatom. The cysteine attacks the methyl group displacing the
guanine in a concerted reaction.

The direct transfer of the methyl group from the DNA to the cysteine will
be investigated by determining whether all three hydrogens are transferred
with the methyl group and by determining the stereochemistry of the methyl
transfer. The activation of guanine as a leaving group by protonation of a
nitrogen on the nucleobase will be probed by synthesizing and then reacting
O6-methyldeazaguanine substrate analogs with AGT. Low reactivity of a
particular O6-methyldeazaguanine substrate will implicate that nitrogen as
a proton acceptor. To determine if AGT activates guanine as a leaving
group by protonation of the oxygen at the 6-position on the nucleoside,
S6-methyl-6-thioguanine and Se6-methyl-6-selenoguanine substrate analogs
will be synthesized and reacted with AGT. Analysis of the kinetic
parameters will indicate whether the protein protonates the oxygen at the
6-position. The protonation state of the cysteine residue will be probed
by investigating the pH profile of reactivity of AGT with the substrates
and with iodoacetamide. The identity of possible basic amino acids in the
active site of AGT which could deprotonate the cysteine will be
investigated by synthesizing and then reacting AGT with active
site-directed alkylating agents.
StatusFinished
Effective start/end date7/1/926/30/93

Funding

  • National Cancer Institute

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