Download The Rad50 molecular hook: a novel structure underlying Mre11 co

Manzan al., 2003
Supplementary Material
0
ATP
+
ATP g
MlaA
Comple
x
Free DNA
(DNA binding)Supplementary Figure 1: ATP dependent DNA binding. Electrophoretic
mobility shift assays of labeled dsDNA 20-mers +/- ATP show that DNA binding of MlaA
is not strongly affected by ATP-binding. Increasing concentrations of MlaA (0, 0.25, 0.5, 1,
2 g) were incubated with 50 fmol of ds oligonucleotide with one strand labeled at the 5
end in 25 mM HEPES, pH 8.0, 50 mM NaCl, 1 mM DTT, 5 mM MgCl2, 10% glycerol, ±1
mM ATP as indicated. Reactions were incubated for 15 minutes at room temperature and
loaded on a 6% polyacrylamide gel in 1X TBE. Complexes were separated and the gel
dried and visualized by phosphoimager..
1
Manzan al., 2003
A
B
b
uf
fe
r
9
00
C
b
uf
fe
r
b
-ATP
+ATP
+ATP
uf
µM
µM
fe µM
MlaA
MlaA
MlaA
r 0. 0. 0. 0. 0. 0. 0. 0. 1 2 4 8
+ 1 2 4 8 1 2 4 8
A
T
P
(Helicase Assays)Supplementary Figure 2: Helicase activity assays. a) MlaA (8 and 16
M) was incubated in 25 mM HEPES, pH 8.0, 50 mM NaCl, 1 mM DTT, 5 mM MgCl 2 and
1 mM ATP with 200 fmol of the indicated Y-substrates at 50C for 15 minutes. Reactions
were terminated by the addition of 3X stop dye (50 mM EDTA, 40% glycerol, 0.9% SDS,
0.1% bromophenol blue, 0.1% xylene cyanol.
Proteinase K was added to a final
concentration of 10 µg/ml and incubated at 37C for ten minutes. Reactions were separated
on a 6% polyacrylamide gel in 1 X TBE, dried and visualized with a phosphoimager.
b) To test for helicase activity of Holliday-junctions, MlaA (indicated concentrations) was
incubated with Holliday junction substrate (gel purified, 50 fmol) in the presence of excess
unlabelled scavenger oligonucleotide Hol1 (supplement Table1) for 30 min at 600C in
2
Manzan al., 2003
50mM Tris 7.5, 100 mM NaCl, 5mM MgCl2, +/- 100 mM ATP. 1% SDS and 10 µg/ml
proteinase K were added and the reaction was incubated at 370C for another 10 min to digest
MlaA. The DNA was resolved by 10% native PAGE in Tris-Borate buffer. In the control
lane (left), the Holliday-junction substrate was incubated at 900C to completely denature the
DNA. The data show no evidence for significant helicase activity. However, it must be noted
that for technical reasons we assayed the MlaA helicase activity appr 400C below
physiological temperatures of P. abyssii (growth temperature above 900C). Thus, we cannot
rule out that MlaA displays significant helicase activity at physiological temperatures.
3
Manzan al., 2003
Supplementary Table 1: Oligonucleotides used for filter binding and helicase assays. Hol1 was
end labeled with 32P. Holliday-junctions (Hol1,2,3,4) and Y-substrates (Hol1,4) were assembled
by mixing stoichiometric amounts of oligos in TBE, followed by slow cooling from 90 0C to 40C
overnight. Products were separated on a 10% non-denaturing polyacrylamide gel. Desired
species were excised, eluted from the gel slices overnight in TBE and used for assays. For
single-strand assays, we used Hol1.
Oligo
Sequence
Hol1
GGCGACGTGATCACCAGATGATGCTAGATGCTTTCCGAAGAGAGAGC
Hol2
GCTCTCTCTTCGGAAAGCATCTAGCATCCTGTCAGCTGCATGGAACG
Hol3
CGTTCCATGCAGCTGACAGGATGCTAGTCAAGGCGAACTGCTAACGG
Hol4
CCGTTAGCAGTTCGCCTTGACTAGCATCATCTGGTGATCACGTCGCC
4