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MICHAEL D. WYATT, Ph.D.
Connor EE, Wilson JJ, Wyatt MD.
Effects of substrate specificity on initiating the base
excision repair of N-methylpurines by variant human
3-methyladenine DNA glycosylases. Chem Res Toxicol. 2005
Jan;18(1):87-94
The human 3-methyladenine (AAG, ANPG,
MPG) DNA glycosylase excises alkylated purines from DNA. In
previous studies, we determined the importance of an active
site amino acid (asparagine 169) in the recognition of
substrates by AAG. In this study, we characterize the
consequences of expressing the AAG variants bearing amino
acid substitutions at position 169 in Saccharomyces
cerevisiae that lack endogenous 3-methyladenine DNA
glycosylase. Survival, mutation induction, and DNA double
strand break formation were determined in response to methyl
methanesulfonate. The ability of purified wild-type and AAG
variants to remove 3-methyladenine and 7-methylguanine, the
two most abundant adducts produced by methyl
methanesulfonate, was also determined. The N169D AAG variant
displayed a approximately 100-fold lower activity for
3-methyladenine as compared to wild-type and did not
detectably remove 7-methylguanine. When expressed in S.
cerevisiae, the N169D variant provided better protection
against methyl methanesulfonate toxicity than wild-type.
Fewer strand breaks in vivo were also seen in the presence
of the N169D variant following MMS exposure. In contrast,
the N169A and N169S AAG variants displayed approximately
30-fold lower activity for 3-methyladenine and
7-methylguanine. Expression of the N169A and N169S AAG
variants in S. cerevisiae during methyl methanesulfonate
exposure resulted in greater sensitivity, greater mutation
induction following MMS exposure, and more strand breaks in
vivo. Strand breaks seen in S. cerevisiae that express
wild-type AAG or the N169 variants were resolved to varying
extents during recovery. In contrast, strand breaks formed
in S. cerevisiae that expressed a catalytically inactive AAG
variant were not resolved during the recovery times
examined. Taken together, the results provide evidence that
3-methyladenine adducts not repaired by base excision repair
cause double strand breaks that are not rapidly resolved.
Evidence is also provided that the BER intermediates
resulting from excision of 7-methylguanine by wild-type AAG
contributes to the mutagenicity and cytotoxicity of
alkylating agents.
Hitchcock TM, Dong L, Connor EE, Meira
LB, Samson LD, Wyatt MD, Cao W. Oxanine DNA glycosylase
activity from Mammalian alkyladenine glycosylase. J Biol
Chem. 2004 Sep 10;279(37):38177-83
Oxanine (Oxa) is a deaminated base lesion
derived from guanine in which the N(1)-nitrogen is
substituted by oxygen. This work reports the mutagenicity of
oxanine as well as oxanine DNA glycosylase (ODG) activities
in mammalian systems. Using human DNA polymerase beta,
deoxyoxanosine triphosphate is only incorporated opposite
cytosine (Cyt). When an oxanine base is in a DNA template,
Cyt is efficiently incorporated opposite the template
oxanine; however, adenine and thymine are also incorporated
opposite Oxa with an efficiency approximately 80% of a Cyt/Oxa
(C/O) base pair. Guanine is incorporated opposite Oxa with
the least efficiency, 16% compared with cytosine. ODG
activity was detected in several mammalian cell extracts.
Among the known human DNA glycosylases tested, human
alkyladenine glycosylase (AAG) shows ODG activity, whereas
hOGG1, hNEIL1, or hNEIL2 did not. ODG activity was detected
in spleen cell extracts of wild type age-matched mice, but
little activity was observed in that of Aag knock-out mice,
confirming that the ODG activity is intrinsic to AAG. Human
AAG can excise Oxa from all four Oxa-containing
double-stranded base pairs, Cyt/Oxa, Thy/Oxa, Ade/Oxa, and
Gua/Oxa, with no preference to base pairing. Surprisingly,
AAG can remove Oxa from single-stranded Oxa-containing DNA
as well. Indeed, AAG can also remove 1,N(6)-ethenoadenine
from single-stranded DNA. This study extends the deaminated
base glycosylase activities of AAG to oxanine; thus, AAG is
a mammalian enzyme that can act on all three purine
deamination bases, hypoxanthine, xanthine, and oxanine.
Li L, Berger SH, Wyatt MD. Involvement
of base excision repair in response to therapy targeted at
thymidylate synthase. Mol Cancer Ther. 2004 Jun;3(6):747-53
Thymidylate synthase (TS) is an important
target of several classes of chemotherapeutic agents.
Although the precise mechanism of cytotoxicity in
thymidylate deprivation remains obscure, uracil
misincorporation and DNA strand breaks are recognized as
important events during thymidylate deprivation. Base
excision repair (BER) plays a primary role in removing
damaged or modified bases from the genome, including uracil.
Because of uracil misincorporation, BER is hypothesized to
play a role in the cellular response to thymidylate
deprivation. In this study, we used murine embryo
fibroblasts wild-type or homozygous null for DNA polymerase
beta (beta-pol), which plays a central role in BER. We found
that, compared with wild-type, beta-pol null cells were
resistant to the toxic effects of raltitrexed (Tomudex,
ZD1694), a folate inhibitor of TS. There was little
difference in TS levels or in TS-ligand complex formation
between the cell lines. Furthermore, cells deficient in
XRCC1, a scaffold protein for the final steps of BER, were
also modestly resistant to raltitrexed compared with
XRCC1-proficient cells. Cell cycle analysis revealed that
the responses of the wild-type and beta-pol null cells were
similar during drug exposure. However, following drug
removal, the beta-pol null cells appeared to resume cell
cycle progression more rapidly than the wild-type cells. The
results suggest that BER plays a role in modulating the
toxic effects of TS inhibitors, and that this role occurs
during recovery from TS inhibition.
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