Table 1 Equilibrium dissociation constants for Atl1–ODN complexes
ODN sequence
KD (nM)
50-SIMA-d(GCCATG(MeG)CTAGTA)a
50-SIMA-d(GCCATG(pobG)CTAGTA)a
50-SIMA-d(GCCATG(5)CTAGTA)
50-SIMA-d(GCCATGGCTAGTA)a
2.4 ꢁ 1.2
1.1 ꢁ 0.02
430 ꢁ 41
740 ꢁ 91
a
Data taken from ref. 18.
analogue of O6-methyl-20-deoxyguanosine. Our data shows
that the anti orientation of the alkyl group appears to block
repair by MGMT and adversely affects binding by Atl1. This
lends further support to the suggestion that MGMT repairs
the syn conformation of O6-alkylguanines and that analogues
which have higher amounts of the anti conformation are
repaired less effectively. In common with MGMT, Atl1 also
shows preferential recognition of the syn conformation.
This work was supported by studentships from PTDF Nigeria
(KA), EPSRC (MKA), BBSRC (OJW), Government of Thailand
(PS) and funding from Cancer Research UK (GPM).
Fig. 5 Structures of compounds 15–18.
the ODN, followed by the addition of excess DNA containing
tritium-labelled O6-methylguanine.6,14 Since the MGMT reaction
is irreversible the residual protein activity can be quantified by
measuring radioactivity transferred to the protein, allowing IC50
values to be determined. The MeG-containing ODN exhibited an
IC50 value of 45 nM. While there was insufficient MGMT
inactivation to determine an IC50 for both the MeG–ODN and
ODN1, an extrapolation of the IC20 (inactivation of 20% of the
MGMT used in the assay) gave values of 0.27 nM for the MeG
ODN and 4000 nM for ODN-1 (ESIy). Thus the former was
around 15 000 times more potent than the latter. Furthermore we
saw no evidence for complex formation between MGMT and
DNA containing 5 following analysis by non-denaturing poly-
acrylamide gel electrophoresis (data not shown), suggesting that
MGMT shows very poor recognition of the base 5.
Notes and references
1 P. F. Swann, Mutat. Res., 1990, 233, 81–94.
2 A. E. Pegg, Mutat. Res., 2000, 462, 83–100.
3 A. E. Pegg, Chem. Res. Toxicol., 2011, 24, 618–639.
4 R. Coulter, M. Blandino, J. M. Tomlinson, G. T. Pauly,
M. Krajewska, R. C. Moschel, L. A. Peterson, A. E. Pegg and
T. E. Spratt, Chem. Res. Toxicol., 2007, 20, 1966–1971.
5 H. Zang, Q. Fang, A. E. Pegg and F. P. Guengerich, J. Biol.
Chem., 2005, 280, 30873–30881.
6 T. Shibata, N. Glynn, T. B. McMurry, R. S. McElhinney, G. P.
Margison and D. M. Williams, Nucleic Acids Res., 2006, 34, 1884–1891.
7 R. S. Mijal, S. Kanugula, C. C. Vu, Q. Fang, A. E. Pegg and
L. A. Peterson, Cancer Res., 2006, 66, 4968–4974.
Recently a family of AGT-related proteins known as alkyl-
transferase-like (ATL) proteins have been discovered in which
the active site Cys is replaced typically by Trp or Ala.15,16 ATL
proteins are consequently catalytically inactive and in organisms
such as S.pombe that lack an AGT protein, the ATL protein
(Atl1) recruits nucleotide excision repair proteins to repair
O6-alkylguanines.17 Structural data of Atl1–DNA complexes15,17
reveal many of the features of MGMT–DNA complexes: The
target base is flipped into the active site and the alkyl group adopts
the syn conformation. However, the hydrophobic binding pocket
for the alkyl group is much larger than that found in MGMT. We
were also interested to assess the recognition of the conformation-
ally-locked analogue 5 by Atl1. Titration of native wild-type Atl1
protein into a solution containing SIMA-labelled ODNs resulted in
a concentration-dependent decrease in fluorescence from which we
derived an equilibrium dissociation constant (Table 1 and ESIy).18
Whilst Atl1 binds to natural DNA (unmodified G-containing
ODN) typically ODNs containing O6-alkylguanines such as
MeG and pobG are bound almost three orders of magnitude
more tightly (Table 1). In contrast, Atl1 has approximately a
2-fold higher affinity for ODN-2 (containing 5) compared to
the natural sequence and an affinity that is dramatically
decreased relative to ODNs containing MeG or pobG. This
highlights the requirement for O6-alkylguanine-containing
substrates to be able to adopt the syn conformation of the
damaged base for effective binding by Atl1.
8 D. S. Daniels, T. T. Woo, K. X. Luu, D. M. Noll, N. D. Clarke,
A. E. Pegg and J. A. Tainer, Nat. Struct. Mol. Biol., 2004, 11, 714–720.
9 Z. Kazimierczuk, H. B. Cottam, G. R. Revankar and R. K. Robins,
J. Am. Chem. Soc., 1984, 106, 6379–6382.
10 F. Seela, B. Westermann and U. Bindig, J. Chem. Soc., Perkin
Trans. 1, 1988, 697–702.
11 D. M. Hammond, D. Edmont, A. R. Hornillo-Araujo and
D. M. Williams, Org. Biomol. Chem., 2003, 1, 4166–4172.
12 A. J. Hubbard, A. S. Jones and R. T. Walker, Nucleic Acids Res.,
1984, 12, 6827–6837.
13 F. Seela and H. Driller, Nucleosides, Nucleotides Nucleic Acids,
1989, 8, 1–21.
14 A. J. Watson and G. P. Margison, in Methods in Molecular
Biology, ed. P. Vaughan, Humana Press, 2000, pp. 49–61.
15 J. L. Tubbs, V. Latypov, S. Kanugula, A. Butt, M. Melikishvili,
R. Kraehenbuehl, O. Fleck, A. Marriott, A. J. Watson,
B. Verbeek, G. McGown, M. Thorncroft, M. F. Santibanez-Koref,
C. Millington, A. S. Arvai, M. D. Kroeger, L. A. Peterson,
D. M. Williams, M. G. Fried, G. P. Margison, A. E. Pegg and
J. A. Tainer, Nature, 2009, 459, 808–813.
16 G. P. Margison, A. Butt, S. J. Pearson, S. Wharton, A. J. Watson,
A. Marriott, C. M. P. F. Caetano, J. J. Hollins, N. Rukazenkova,
G. Begum and M. F. Santibanez-Koref, DNA Repair, 2007, 6, 1222.
17 V. F. Latypov, J. L. Tubbs, A. J. Watson, A. S. Marriott,
G. McGown, M. Thorncroft, O. J. Wilkinson, P. Senthong,
A. Butt, A. S. Arvai, C. L. Millington, A. C. Povey,
D. M. Williams, M. F. Santibanez-Koref, J. A. Tainer and
G. P. Margison, Mol. Cell, 2012, 47, 50–60.
18 O. J. Wilkinson, V. Latypov, J. L. Tubbs, C. L. Millington,
R. Morita, H. Blackburn, A. Marriott, G. McGown,
M. Thorncroft, A. J. Watson, B. A. Connolly, J. A. Grasby,
R. Masui, C. A. Hunter, J. A. Tainer, G. P. Margison and
D. M. Williams, Proc. Natl. Acad. Sci. U. S. A., DOI: 10.1073/
pnas.1209451109.
In summary we have described the synthesis of DNA
containing the first example of a conformationally-locked anti
c
11216 Chem. Commun., 2012, 48, 11214–11216
This journal is The Royal Society of Chemistry 2012