DNA polymerase selectivity:
sugar interactions monitored with high fidelity nucleotides
Daniel Summerer, Christian Schneider and Andreas Marx*
Kekulé-Institut für Organische Chemie und Biochemie, Universität Bonn, Gerhard-Domagk-Str. 1, D-53121
Bonn
a.marx@uni-bonn.de
The essential prerequisite of any organism is to keep its genome intact and to accurately
duplicate it before cell division. All DNA synthesis required for DNA repair, recombination
and replication depends on the ability of DNA polymerases to recognise the template and
correctly insert the complementary nucleotide. In current concepts of DNA replication,
fidelity is primarily achieved by DNA polymerases through editing of base pair geometry. [1]
This model is further supported by crystal structures of DNA polymerases which suggest that
DNA polymerases build a tight nucleotide binding pocket to accommodate Watson-Crick
base pairs while misinsertion is prevented due to steric constrains.
Since most functional studies focused on nucleobase recognition processes,[1] little is
known about the impact of DNA polymerase interactions with the 2'-deoxyribose moiety and
their participations in processes which contribute to fidelity. We developed a functional
strategy to monitor steric constraints in DNA polymerases within the nucleotide binding
pocket acting on the sugar moiety of an incoming nucleoside triphosphate employing
modified nucleotide probes.[2] To minimize undesired additional effects caused by the probe,
we chemically introduced the modification at the 2'-deoxyribose moiety without interfering
with Watson-Crick hydrogen bonding patterns (Fig. 1). Through application of these probes
in functional studies of DNA polymerases we should be able to monitor steric constrains in
the nucleotide binding pocket of DNA polymerases uncoupled from effects caused by
hydrogen bonding, base pairing and base stacking.
O
O
N
O
PPPO
O
N
O
H
OH
O
R: CH 3
NH
NH
NH
O
O
O
O P O P O P O
O
O
O
O
PPPO
N
O
R
OH
OH
TTP
TRTP
O
CH 3
CH 3
CH 3
CH 3
H3C
Figure 1: Design of modified tymidine triphosphates as steric probes. The
modifications were introduced in a multistep synthesis starting from thymidine.
We performed qualitative and quantitative[3] investigations of the Klenow Fragment (KF) of
E. coli DNA polymerase I (exo- mutant) using a in vitro replication assay. Our results exhibit
that KF- incorporates the smallest thymidine analog TMeTP and TEtTP opposite a WatsonCrick base with virtually the same efficiency than unmodified TTP (Fig. 2).
Primer (24mer)
Template (36mer
T*TP
5‘----ACA
3‘----TGTACT-----
b)
efficiency (Vmax/KM)
a)
25
24
1000000
10000
100
1
[M]: 0
R:
1
50
H
1
50 1
Me
50 1
Et
50 1
iPr
50
H
iBu
Me Et iPr iBu
Figure 2: a) dNTP insertion by the Klenow fragment (exo-) of E. coli DNA
polymerase I. b) Steady-state analyses for canonical nucleoside triphosphate
insertion.
We next investigated the ability of KF- to catalyze non-Watson-Crick nucleobase pair
formation. We focused on TMeTP and TEtTP since only TMeTP and TEtTP are inserted as
efficiently as TTP by KF- in canonical base pair formation (see Fig. 2) and thus, should be
ideally suited to monitor differential enzyme interactions between insertion and misinsertion
events. Strikingly, TMeTP and TEtTP exhibit dramatically decreased misinsertion efficiency:
steady-state kinetic analyses revealed an approximately 100-fold decrease in misinsertion
efficiency using the 4'-alkylated probes TMeTP and TEtTP compared to TTP (Fig 3).
Primer (24mer)
Template (36mer
T*TP
5‘----ACA
3‘----TGTGCT-----
25
24
[M]: 0
1
50
dCTP
50 500
50 500
TTP
Me
50
1000
b)
efficiency (Vmax/KM)
a)
100
10
1
500
Et
H
Me
Et
Figure 3: a) dNTP misinsertion by the Klenow fragment (exo-) of E. coli DNA
polymerase I. b) Steady-state analyses for non-canonical nucleoside triphosphate
insertion .
Further experiments also showed that the analogues TMeTP and TEtTP were not as well
inserted opposite T or C as was unmodified TTP (see Fig. 4). These results clearly exhibit that
nucleotide insertion selectivity is increased by substitution of the 4'-hydrogen at the sugar
with bulkier alkyl groups.
Primer (24mer)
Template (36mer
T*TP
5‘----ACA
3‘----TGTCCT-----
Primer (24mer)
Template (36mer
25
25
24
24
[M]: 0
50 500
TTP
50 500 50
Me
500
[M]: 0
Et
50
500
TTP
T*TP
5‘----ACA
3‘----TGTTCT-----
50 500 50
Me
500
Et
Figure 4: dNTP misinsertion by the Klenow fragment (exo-) of E. coli DNA
polymerase I opposite template C and T.
In conclusion, we were able to show that selectivity of nucleotide insertion by a DNA
polymerase can be significantly increased by modified sugar moieties. Our results strongly
implicate the involvement of differential DNA polymerase interactions with the sugar in
processes that contribute to the fidelity of DNA synthesis. Furthermore, our studies provide a
new functional and general method to monitor steric constraints in nucleotide binding pockets
of DNA polymerases. Further analyses with such steric probes, both at the functional and
structural level should reveal more insights into mechanisms of DNA polymerase selectivity.
[1] Recent reviews and commentaries: a) T. A. Kunkel, K. Bebenek, Annu. Rev. Biochem.
2000, 69, 497–529; b) E. T. Kool, J. C. Morales, K. M. Guckian, Angew. Chem. Int. Ed.
2000, 39, 991–1009; c) T. A. Kunkel, S. H. Wilson, Nature Struc. Biol. 1998, 5, 95–99;
d) U. Diederichsen, Angew. Chem. Int. Ed. 1998, 37, 1655–1657; e) M. F. Goodman,
Proc. Natl. Acad. Sci. USA 1997, 94, 10493–10495.
[2] D. Summerer, A. Marx, Angew. Chem. Int. Ed. in press.
[3] S. Creighton, L. B. Bloom, M. F. Goodman, Methods Enzymol. 1995, 262, 232–256.