Figure 2.
Figure 2. WRN-exo metal-ion dependence and structural analyses.
(a) Nuclease activity assays containing WRN-exo (50 pmol) and
radiolabeled DNA substrate were incubated for 30 min with either
no metal (control; lane 1) or the noted divalent cation(s).
WRN-exo 3' arrow
5' dsDNA nuclease activity is supported by Mg^2+ or Mn^2+ ions,
but not by Eu^2+ or in the absence of divalent cations. Addition
of Eu^2+ inhibits nuclease activity in the presence of equimolar
Mg^2+ or Mn^2+ ions. The DNA digestion pattern with equimolar
Mg^2+ and Mn^2+ is indistinguishable from that of Mn^2+ alone.
(b) Two Mn^2+ ions (purple) are chelated in the WRN active site
in the absence of DNA; dashed magenta lines denote metal-ion
bonds, with distances labeled. The inner metal ion, M[A], is
directly coordinated by Asp82, Glu84 and Asp216, and the outer
metal ion, M[B], directly ligates one side chain, Asp82, that
bridges the two metal ions. Asp143 has an indirect interaction
with the M[B] metal ion via two water molecules. (c) The WRN
active site also accommodates two of the larger lanthanide Eu^3+
ions (blue) in the absence of substrate. Dashed blue lines
denote metal-ion bonds. (d) Overlay of WRN Mn^2+ and Eu^3+
metal-ion complex structures, colored as in b and c.
Incorporation of Eu^3+ metal ions at sites M[A] and M[B] does
not cause appreciable changes in the WRN active site.
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5' dsDNA nuclease activity is supported by Mg^2+ or Mn^2+ ions,
but not by Eu^2+ or in the absence of divalent cations. Addition
of Eu^2+ inhibits nuclease activity in the presence of equimolar
Mg^2+ or Mn^2+ ions. The DNA digestion pattern with equimolar
Mg^2+ and Mn^2+ is indistinguishable from that of Mn^2+ alone.
(b) Two Mn^2+ ions (purple) are chelated in the WRN active site
in the absence of DNA; dashed magenta lines denote metal-ion
bonds, with distances labeled. The inner metal ion, M[A], is
directly coordinated by Asp82, Glu84 and Asp216, and the outer
metal ion, M[B], directly ligates one side chain, Asp82, that
bridges the two metal ions. Asp143 has an indirect interaction
with the M[B] metal ion via two water molecules. (c) The WRN
active site also accommodates two of the larger lanthanide Eu^3+
ions (blue) in the absence of substrate. Dashed blue lines
denote metal-ion bonds. (d) Overlay of WRN Mn^2+ and Eu^3+
metal-ion complex structures, colored as in b and c.
Incorporation of Eu^3+ metal ions at sites M[A] and M[B] does
not cause appreciable changes in the WRN active site.