Initiating the DNA ba
se exci
sion repair pathway, DNA glyco
syla
se
s find and hydrolytically exci
sedamaged ba
se
s from DNA. While
some DNA glyco
syla
se
s exhibit narrow
specificity, other
s remove multipleform
s of damage. Human thymine DNA glyco
syla
se (hTDG) cleave
s thymine from mutagenic G·T mi
spair
s,recognize
s many additional le
sion
s, and ha
s a
strong preference for nucleoba
se
s paired with guaninerather than adenine. Yet, hTDG avoid
s cyto
sine, de
spite the million-fold exce
ss of normal G·C pair
s overG·T mi
spair
s. The mechani
sm of thi
s remarkable and e
ssential
specificity ha
s remained ob
scure. Here,we examine the po
ssibility that hTDG
specificity depend
s on the
stability of the
sci
ssile ba
se-
sugar bondby determining the maximal activity (
kmax) again
st a
serie
s of nucleoba
se
s with varying leaving-group ability.We find that hTDG remove
s 5-fluorouracil 78-fold fa
ster than uracil, and 5-chlorouracil, 572-fold fa
sterthan thymine, difference
s that can be attributed predominantly to leaving-group ability. Moreover, hTDGreadily exci
se
s cyto
sine analogue
s with improved leaving ability, including 5-fluorocyto
sine, 5-bromocyto
sine,and 5-hydroxycyto
sine, indicating that cyto
sine ha
s acce
ss to the active
site. A plot of log(
kmax) ver
su
sleaving-group p
Ka reveal
s a Br
![](/image<font color=)
s/entitie
s/o
sla
sh.gif">n
sted-type linear free energy relation
ship with a large negative
slope of
![](/image<font color=)
s/gifchar
s/beta2.gif" BORDER=0 ALIGN="middle">
lg= -1.6 ± 0.2, con
si
stent with a highly di
ssociative reaction mechani
sm. Further, we find that the hydrophobicactive
site of hTDG contribute
s to it
s specificity by enhancing the inherent difference
s in
sub
strate reactivity.Thu
s, hTDG
specificity depend
s on
N-glyco
sidic bond
stability, and the di
scrimination again
st cyto
sine i
sdue largely to it
s very poor leaving ability rather than it
s exclu
sion from the active
site.