Figure 5.

Single-turnover kinetic experiments on free DNA (U⁺⁴⁴) and NCPs containing T→U substitutions at three single sites (U⁺²²N1, U⁻³²N1 and U⁺⁴⁴N1). (a) Observed rate constants (k_obs^sto) for reaction of 20 nM free 601 DNA containing a uracil at site U⁺⁴⁴ with various concentrations of UNG. The insert shows the kinetic time course with 0.4 µM UNG. (b) Observed rate constants (k_obs^sto) for reaction of 20 nM NCP containing a uracil at site U⁺²²N1 with various concentrations of UNG. The insert shows the kinetic time course with 1.3 µM UNG. (c) Time course (log scale) for reaction of 20 nM NCP containing uracil at site U⁻³²N1 with 0.2 µM UNG. (d) Time course (log scale) for reaction of 20 nM NCP containing uracil at site U⁺⁴⁴N1 with 0.2 µM UNG. In panels b-c the graphic inserts display the structural properties of each NCP site. The uracils are in red, the histones are in purple and the DNA is in grey. (e, f) Absence of hUNG concentration dependence for the observed rate constants for single-turnover excision of uracil from nucleosome sites U⁺⁴⁴N1 and U⁻³²N1.