Kinetic Analysis of an Efficient DNA-dependent TNA
Polymerase
Supplementary Material
Allen Horhota,2 Keyong Zou, 1 Justin K. Ichida, 1 Biao Yu,3 Larry W. McLaughlin,2 Jack W.
Szostak1 and John C. Chaput1†*
1
Howard Hughes Medical Institute, and Department of Molecular Biology, Massachusetts General Hospital, Boston,
MA 02114
2
3
Department of Chemistry, Boston College, Chestnut Hill, MA 02467
State Key Laboratory of Bioorganic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry,
Chinese Academy of Sciences, Shanghai 200032, China
To whom correspondence should be addressed. Telephone: (480) 727-0392 email: john.
chaput@asu.edu
Steady State Reactions Experimental Methods.
α-L-Threofuranosyl nucleoside triphosphates were synthesized as decribed by Zou et. al1.
Standing start single nucleotide incorporation kinetic experiments were conducted according to
the method described by Goodman2. Reactions were carried out in 20 µl of 20 mM Tris-HCl, 10
mM KCl, 10 mM (NH4)2SO4, 20 mM MgSO4, 0.1% Triton X-100, 0.25 µg/µl BSA, 100 µM
DTT with 0.05 units of Therminator DNA Polymerase (New England Biolabs) with or without
the addition of freshly prepared MnCl2. Reaction solutions were preheated at 55° C and initiated
by the addition of 10 µl of 2X NTP to an equal volume of solution containing both polymerase
and P32 labeled primer template complex. Reaction times were designed to limit total primer
extension to 20% (typically 1-3 min) at which time the reactions were quenched by the addition
of stop buffer containing 8M urea, 100 mM EDTA, and 1X TBE buffer. Samples were analyzed
using 20% denaturing PAGE followed by phosphorimaging and quantitation on a Bio-Rad
Molecular Imager FX. The insertion kinetic parameters of Vmax and Km were determined using
Hanes-Woolf plots to give an overall insertion efficiency for each NTP.
References
1. Zou, K.; Horhota, A.; Yu, B.; Szostak, J.W.; McLaughlin, L.W. Org. Lett. 2005, In press.
2. (a) Boosalis, M. S.; Petruska, J.; Goodman, M. F. J. Biol. Chem. 1987, 262, 14689-14696. (b)
Goodman, M. F.; Creighton, S.; Bloom, L. B.; Petruska, J. Crit. Rev. Biochem. Mol. Biol.
1993, 28, 83-126.
A
0.14
0.12
0.1
0.08
R2 = 0.9995
0.06
0.04
0.02
0
0
0.5
1
1.5
2
2.5
0.25
0.2
0.15
R2 = 0.9979
0.1
0.05
0
0
3
1
2
C
tGTP Mg2+
4
5
0.1
0.08
tCTP Mg2+
6
R2 = 0.9936
0.06
0.04
0.02
0
D
0.3
[S]/V (uM/% Product
per minute)
0.12
[S]/V (uM/% Product
per minute)
3
[S] (uM)
[S] (uM)
0.25
0.2
R2 = 0.9987
0.15
0.1
0.05
0
0
0.5
1
1.5
2
2.5
3
[S] (uM)
0
1
2
3
4
5
[S] (uM)
30
E
25
% Product/Min
B
tTTP Mg2+
[S]/V (uM/% Product
per minute)
[S]/V (uM/% Product
per minute)
tDTP Mg2+
20
tDTP Mg
tTTP Mg
15
tGTP Mg
tCTP Mg
10
5
0
0
2
4
6
8
10
12
14
Triphosphate Concentration (uM)
Fig. 1 (supplementary). A-D are Steady-State Hanes-Woolf plots for the incorporation of tNTPs by
Therminator DNA Polymerase in the presence of Mg2+ cations from which Vmax and Km values were
determined to yield an overall insertion efficiency. A. tDTP opposite T. B. tTTP opposite A. C. tGTP
opposite C. D. tCTP opposite G. E. Michaelis-Menten nonlinear regression plot showing all four
tNTPs insertion velocities relative to substrate concentration.
6
A
[S]/V (uM/% Product
per minute)
0.3
0.25
0.25
0.2
R2 = 0.9996
0.15
0.1
0.05
0
0
1
2
3
4
5
0.2
0.15
R2 = 0.9928
0.1
0.05
0
6
0
1
2
3
[S] (uM)
C
5
6
0.3
0.25
0.2
0.15
D
tCTP Mn2+
[S]/V (uM/% Product
per minute)
0.3
[S]/V (uM/% Product
per minute)
4
[S] (uM)
tGTP Mn2+
2
R = 0.9945
0.1
0.05
0
0.25
0.2
0.15
2
R = 0.9888
0.1
0.05
0
0
1
2
3
4
5
6
[S] (uM)
0
1
2
3
4
5
6
[S] (uM)
E
30
25
% Product/Min
B
tTTP Mn2+
[S]/V (uM/% Product
per minute)
tDTP Mn2+
20
tDTP Mn
tTTP Mn
15
tGTP Mn
tCTP Mn
10
5
0
0
2
4
6
8
10
12
14
Triphosphate Concentration (uM)
Fig. 2 (supplementary). A-D are Steady-State Hanes-Woolf plots for the incorporation of tNTPs by
Therminator DNA Polymerase in the presence of Mn2+ cations from which Vmax and Km values were
determined to yield an overall insertion efficiency. A. tDTP opposite T. B. tTTP opposite A. C. tGTP
opposite C. D. tCTP opposite G. E. Michealis-Menten nonlinear regression plot showing all four
tNTPs insertion velocities relative to substrate concentration.
A
tDTP
3.5
[S]/V (uM/% Product
per minute)
[S]/V (uM/% Product
per minute)
3.5
3
2.5
R2 = 0.9913
2
1.5
1
0.5
3
2.5
R2 = 0.9575
2
1.5
1
0.5
0
0
-1
1
3
5
7
9
11
13
0
15
5
10
C
[S]/V (uM/% Product
per minute)
4
3.5
3.5
3
2.5
2
R = 0.9909
2
1.5
1
0.5
3
2.5
R2 = 0.9801
2
1.5
1
0.5
0
0
1
3
5
7
9
11
13
15
-1
1
3
5
[S] (uM)
7
9
11
13
E
2+
F
1.80
16
1.60
14
% Product/Min
1.40
2
R = 0.987
1.20
1.00
0.80
0.60
0.40
0.20
12
tTTP
10
tGTP Mn2+
8
tCTP
tDTP
6
tGTP
4
2
0.00
1
3
5
7
[S] (uM)
15
[S] (uM)
tGTP Mn
[S]/V (uM/% Product
per minute)
D
tCTP
[S]/V (uM/%
Product per minute)
tGTP
-1
15
[S] (uM)
[S] (uM)
-1
B
tTTP
9
11
13
15
0
0
5
10
15
20
25
30
Triphosphate Concentration (uM)
Fig. 3 (supplementary). A-E are Steady-State Hanes-Woolf plots for the incorporation of tNTPs by
Deep Vent DNA Polymerase from which Vmax and Km values were determined to yield an overall
insertion efficiency. A. tDTP opposite T. B. tTTP opposite A. C. tGTP opposite C. D. tCTP opposite
G. E. tGTP opposite C with Mn2+ F. Michealis-Menten nonlinear regression plot showing all four
tNTPs insertion velocities relative to substrate concentration.