5 research outputs found
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
Dynamic Equilibria of Short-Range Electrostatic Interactions at Molecular Interfaces of Protein–DNA Complexes
Intermolecular ion pairs (salt bridges)
are crucial for protein–DNA
association. For two protein–DNA complexes, we demonstrate
that the ion pairs of protein side-chain NH<sub>3</sub><sup>+</sup> and DNA phosphate groups undergo dynamic transitions between distinct
states in which the charged moieties are either in direct contact
or separated by water. While the crystal structures of the complexes
show only the solvent-separated ion pair (SIP) state for some interfacial
lysine side chains, our NMR hydrogen-bond scalar coupling data clearly
indicate the presence of the contact ion pair (CIP) state for the
same residues. The 0.6-μs molecular dynamics (MD) simulations
confirm dynamic transitions between the CIP and SIP states. This behavior
is consistent with our NMR order parameters and scalar coupling data
for the lysine side chains. Using the MD trajectories, we also analyze
the free energies of the CIP–SIP equilibria. This work illustrates
the dynamic nature of short-range electrostatic interactions in DNA
recognition by proteins
Residence Times of Molecular Complexes in Solution from NMR Data of Intermolecular Hydrogen-Bond Scalar Coupling
The
residence times of molecular complexes in solution are important
for understanding biomolecular functions and drug actions. We show
that NMR data of intermolecular hydrogen-bond scalar couplings can
yield information on the residence times of molecular complexes in
solution. The molecular exchange of binding partners via the breakage
and reformation of a complex causes self-decoupling of intermolecular
hydrogen-bond scalar couplings, and this self-decoupling effect depends
on the residence time of the complex. For protein–DNA complexes,
we investigated the salt concentration dependence of intermolecular
hydrogen-bond scalar couplings between the protein side-chain <sup>15</sup>N and DNA phosphate <sup>31</sup>P nuclei, from which the
residence times were analyzed. The results were consistent with those
obtained by <sup>15</sup>N<sub><i>z</i></sub>-exchange spectroscopy.
This self-decoupling-based kinetic analysis is unique in that it does
not require any different signatures for the states involved in the
exchange, whereas such conditions are crucial for kinetic analyses
by typical NMR and other methods
Direct Observation of the Ion-Pair Dynamics at a Protein–DNA Interface by NMR Spectroscopy
Ion pairing is one
of the most fundamental chemical interactions
and is essential for molecular recognition by biological macromolecules.
From an experimental standpoint, very little is known to date about
ion-pair dynamics in biological macromolecular systems. Absorption,
infrared, and Raman spectroscopic methods were previously used to
characterize dynamic properties of ion pairs, but these methods can
be applied only to small compounds. Here, using NMR <sup>15</sup>N
relaxation and hydrogen-bond scalar <sup>15</sup>N–<sup>31</sup>P <i>J</i>-couplings (<sup><i>h3</i></sup><i>J</i><sub>NP</sub>), we have investigated the dynamics of the
ion pairs between lysine side-chain NH<sub>3</sub><sup>+</sup> amino
groups and DNA phosphate groups at the molecular interface of the
HoxD9 homeodomain–DNA complex. We have determined the order
parameters and the correlation times for C–N bond rotation
and reorientation of the lysine NH<sub>3</sub><sup>+</sup> groups.
Our data indicate that the NH<sub>3</sub><sup>+</sup> groups in the
intermolecular ion pairs are highly dynamic at the protein–DNA
interface, which should lower the entropic costs for protein–DNA
association. Judging from the C–N bond-rotation correlation
times along with experimental and quantum-chemically derived <sup><i>h3</i></sup><i>J</i><sub>NP</sub> hydrogen-bond
scalar couplings, it seems that breakage of hydrogen bonds in the
ion pairs occurs on a sub-nanosecond time scale. Interestingly, the
oxygen-to-sulfur substitution in a DNA phosphate group was found to
enhance the mobility of the NH<sub>3</sub><sup>+</sup> group in the
intermolecular ion pair. This can partially account for the affinity
enhancement of the protein–DNA association by the oxygen-to-sulfur
substitution, which is a previously observed but poorly understood
phenomenon
Disordered N‑Terminal Domain of Human Uracil DNA Glycosylase (hUNG2) Enhances DNA Translocation
Nuclear
human uracil–DNA glycosylase (hUNG2) initiates base
excision repair (BER) of genomic uracils generated through misincorporation
of dUMP or through deamination of cytosines. Like many human DNA glycosylases,
hUNG2 contains an unstructured N-terminal domain that encodes a nuclear
localization signal, protein binding motifs, and sites for post-translational
modifications. Although the N-terminal domain has minimal effects
on DNA binding and uracil excision kinetics, we report that this domain
enhances the ability of hUNG2 to translocate on DNA chains as compared
to the catalytic domain alone. The enhancement is most pronounced
when physiological ion concentrations and macromolecular crowding
agents are used. These data suggest that crowded conditions in the
human cell nucleus promote the interaction of the N-terminus with
duplex DNA during translocation. The increased contact time with the
DNA chain likely contributes to the ability of hUNG2 to locate densely
spaced uracils that arise during somatic hypermutation and during
fluoropyrimidine chemotherapy