4 research outputs found
Electrostatic Properties of Complexes along a DNA Glycosylase Damage Search Pathway
Human uracil DNA glycosylase (hUNG)
follows an extended reaction
coordinate for locating rare uracil bases in genomic DNA. This process
begins with diffusion-controlled engagement of undamaged DNA, followed
by a damage search step in which the enzyme remains loosely associated
with the DNA chain (translocation), and finally, a recognition step
that allows the enzyme to efficiently bind and excise uracil when
it is encountered. At each step along this coordinate, the enzyme
must form DNA interactions that are highly specialized for either
rapid damage searching or catalysis. Here we make extensive measurements
of hUNG activity as a function of salt concentration to dissect the
thermodynamic, kinetic, and electrostatic properties of key enzyme
states along this reaction coordinate. We find that the interaction
of hUNG with undamaged DNA is electrostatically driven at a physiological
concentration of potassium ions (Δ<i>G</i><sub>elect</sub> = −3.5 ± 0.5 kcal mol<sup>–1</sup>), with only
a small nonelectrostatic contribution (Δ<i>G</i><sub>non</sub> = −2.0 ± 0.2 kcal mol<sup>–1</sup>).
In contrast, the interaction with damaged DNA is dominated by the
nonelectrostatic free energy term (Δ<i>G</i><sub>non</sub> = −7.2 ± 0.1 kcal mol<sup>–1</sup>), yet retains
the nonspecific electrostatic contribution (Δ<i>G</i><sub>elect</sub> = −2.3 ± 0.2 kcal mol<sup>–1</sup>). Stopped-flow kinetic experiments established that the salt sensitivity
of damaged DNA binding originates from a reduction of <i>k</i><sub>on</sub>, while <i>k</i><sub>off</sub> is weakly dependent
on salt. Similar findings were obtained from the salt dependences
of the steady-state kinetic parameters, where the diffusion-controlled <i>k</i><sub>cat</sub>/<i>K</i><sub>m</sub> showed a
salt dependence similar to <i>k</i><sub>on</sub>, while <i>k</i><sub>cat</sub> (limited by product release) was weakly
dependent on salt. Finally, the salt dependence of translocation between
two uracil sites separated by 20 bp in the same DNA chain was indistinguishable
from that of <i>k</i><sub>on</sub>. This result suggests
that the transition-state for translocation over this spacing resembles
that for DNA association from bulk solution and that hUNG escapes
the DNA ion cloud during translocation. These findings provide key
insights into how the ionic environment in cells influences the DNA
damage search pathway
AP-Endonuclease 1 Accelerates Turnover of Human 8‑Oxoguanine DNA Glycosylase by Preventing Retrograde Binding to the Abasic-Site Product
A major
product of oxidative DNA damage is 8-oxoguanine. In humans,
8-oxoguanine DNA glycosylase (hOGG1) facilitates removal of these
lesions, producing an abasic (AP) site in the DNA that is subsequently
incised by AP-endonuclease 1 (APE1). APE1 stimulates turnover of several
glycosylases by accelerating rate-limiting product release. However,
there have been conflicting accounts of whether hOGG1 follows a similar
mechanism. In pre-steady-state kinetic measurements, we found that
addition of APE1 had no effect on the rapid burst phase of 8-oxoguanine
excision by hOGG1 but accelerated steady-state turnover (<i>k</i><sub>cat</sub>) by ∼10-fold. The stimulation by APE1 required
divalent cations, could be detected under multiple-turnover conditions
using limiting concentrations of APE1, did not require flanking DNA
surrounding the hOGG1 lesion site, and occurred efficiently even when
the first 49 residues of APE1’s N-terminus had been deleted.
Stimulation by APE1 does not involve relief from product inhibition
because thymine DNA glycosylase, an enzyme that binds more tightly
to AP sites than hOGG1 does, could not effectively substitute for
APE1. A stimulation mechanism involving stable protein–protein
interactions between free APE1 and hOGG1, or the DNA-bound forms,
was excluded using protein cross-linking assays. The combined results
indicate a mechanism whereby dynamic excursions of hOGG1 from the
AP site allow APE1 to invade the site and rapidly incise the phosphate
backbone. This mechanism, which allows APE1 to access the AP site
without forming specific interactions with the glycosylase, is a simple
and elegant solution to passing along unstable intermediates in base
excision repair