22 research outputs found
Eukaryotic Translesion DNA Synthesis on the Leading and Lagging Strands: Unique Detours around the Same Obstacle
During S-phase, minor DNA damage
may be overcome by DNA damage
tolerance (DDT) pathways that bypass such obstacles, postponing repair
of the offending damage to complete the cell cycle and maintain cell
survival. In translesion DNA synthesis (TLS), specialized DNA polymerases
replicate the damaged DNA, allowing stringent DNA synthesis by a replicative
polymerase to resume beyond the offending damage. Dysregulation of
this DDT pathway in human cells leads to increased mutation rates
that may contribute to the onset of cancer. Furthermore, TLS affords
human cancer cells the ability to counteract chemotherapeutic agents
that elicit cell death by damaging DNA in actively replicating cells.
Currently, it is unclear how this critical pathway unfolds, in particular,
where and when TLS occurs on each template strand. Given the semidiscontinuous
nature of DNA replication, it is likely that TLS on the leading and
lagging strand templates is unique for each strand. Since the discovery
of DDT in the late 1960s, most studies on TLS in eukaryotes have focused
on DNA lesions resulting from ultraviolet (UV) radiation exposure.
In this review, we revisit these and other related studies to dissect
the step-by-step intricacies of this complex process, provide our
current understanding of TLS on leading and lagging strand templates,
and propose testable hypotheses to gain further insights
Replication Protein A Prohibits Diffusion of the PCNA Sliding Clamp along Single-Stranded DNA
The
replicative polymerases cannot accommodate distortions to the
native DNA sequence such as modifications (lesions) to the native
template bases from exposure to reactive metabolites and environmental
mutagens. Consequently, DNA synthesis on an afflicted template abruptly
stops upon encountering these lesions, but the replication fork progresses
onward, exposing long stretches of the damaged template before eventually
stalling. Such arrests may be overcome by translesion DNA synthesis
(TLS) in which specialized TLS polymerases bind to the resident proliferating
cell nuclear antigen (PCNA) and replicate the damaged DNA. Hence,
a critical aspect of TLS is maintaining PCNA at or near a blocked
primer/template (P/T) junction upon uncoupling of fork progression
from DNA synthesis by the replicative polymerases. The single-stranded
DNA (ssDNA) binding protein, replication protein A (RPA), coats the
exposed template and might prohibit diffusion of PCNA along the single-stranded
DNA adjacent to a blocked P/T junction. However, this idea had yet
to be directly tested. We recently developed a unique Cy3-Cy5 Forster
resonance energy transfer (FRET) pair that directly reports on the
occupancy of DNA by PCNA. In this study, we utilized this FRET pair
to directly and continuously monitor the retention of human PCNA at
a blocked P/T junction. Results from extensive steady state and pre-steady
state FRET assays indicate that RPA binds tightly to the ssDNA adjacent
to a blocked P/T junction and restricts PCNA to the upstream duplex
region by physically blocking diffusion of PCNA along ssDNA
Capturing a Sulfenic Acid with Arylboronic Acids and Benzoxaborole
Post-translational redox generation
of cysteine-sulfenic acids
(Cys-SOH) functions as an important reversible regulatory mechanism
for many biological functions, such as signal transduction, balancing
cellular redox states, catalysis, and gene transcription. Herein we
show that arylboronic acids and cyclic benzoxaboroles can form adducts
with sulfenic acids in aqueous medium and that these boron-based compounds
can potentially be used to trap biologically significant sulfenic
acids. As proof of principle we demonstrate that a benzoxaborole can
inhibit the enzyme activity of an iron-containing nitrile hydratase,
which requires a catalytic αCys114-SOH in the active site. The
nature of the adduct and the effect of the boronic acid’s p<i>K</i><sub>a</sub><sup>B</sup> on the stability constant of the
adduct are discussed within
Examination of the Reactivity of Benzoxaboroles and Related Compounds with a <i>cis</i>-Diol
Benzoxaboroles have been emerging as an interesting and
useful
scaffold in drug discovery due to their apparently unique reactivity
toward diols under physiological conditions. In this work, the reaction
of benzoxaborole with the diol-containing, fluorescent dye Alizarin
Red S is probed. Steady-state and presteady-state experiments have
been conducted for the characterization of the reactions over a wide
range of pH. Results indicate that Alizarin Red S reacts with both
the boronic (neutral, trigonal) form as well as the boronate (anionic,
tetrahedral) form of benzoxaborole in a reaction largely analogous
to that previously determined for the simple phenylboronic acid. However,
in certain key aspects, the reactivity of the benzoxaborole was found
to differ from that of simple phenylboronic acid. The structural origin
of these differences has been explored by examination of compounds
related to both benzoxaborole and phenylboronic acid. These results
may be applied to rational drug discovery efforts aimed at expanding
the use of benzoxaboroles in medicine
Elucidation of the Mechanism of the Reaction between Phenylboronic Acid and a Model Diol, Alizarin Red S
In this work, the reaction between phenylboronic acid
and the diol-containing,
fluorescent dye Alizarin Red S (<b>ARS</b>) was probed. Fluorescence
titrations, <sup>11</sup>B NMR measurements, and both pre- and steady-state
kinetic experiments were used for the characterization of this reaction
over a large pH range (4–10.5). It was shown that <b>ARS</b> preferentially reacted with the
boronic (neutral, trigonal) form of phenylboronic acid; however, the
boronate (anionic, tetrahedral) form was also reactive. All in all,
four reactant species were implicated in the formation of four different
adduct species. The rate of a given adduct formation depended on the
combination of the solution pH and the p<i>K</i><sub>a</sub>’s of both <b>ARS</b> and the
arylboronic acid. The reaction was found to proceed in two distinct
kinetic steps with the products and starting materials in facile exchange.
In addition, the elucidation of the mechanism indicated the presence
of two fluorescent products with the structure of the major contributor
differing from what had been cited in the literature
Monitoring the Retention of Human Proliferating Cell Nuclear Antigen at Primer/Template Junctions by Proteins That Bind Single-Stranded DNA
In
humans, proliferating cell nuclear antigen (PCNA) sliding clamps
encircling DNA coordinate various aspects of DNA metabolism throughout
the cell cycle. A critical aspect of this is restricting PCNA to the
vicinity of its DNA target site. For example, PCNA must be maintained
at or near primer/template (P/T) junctions during DNA synthesis. With
a diverse array of cellular factors implicated, many of which interact
with PCNA, DNA, or both, it is unknown how this critical feat is achieved.
Furthermore, current biochemical assays that examine the retention
of PCNA near P/T junctions are inefficient, discontinuous, and qualitative
and significantly deviate from physiologically relevant conditions.
To overcome these challenges and limitations, we recently developed
a novel and convenient Förster resonance energy transfer (FRET)
assay that directly and continuously monitors the retention of human
PCNA at a P/T junction. Here we describe in detail the design, methodology,
interpretation, and limitations of this quantitative FRET assay using
the single-stranded DNA-binding protein, SSB, from <i>Escherichia
coli</i> as an example. This powerful tool is broadly applicable
to any single-stranded DNA-binding protein and may be utilized and/or
expanded upon to dissect DNA metabolic pathways that are dependent
upon PCNA
Ring Structure and Aromatic Substituent Effects on the p<i>K</i><sub>a</sub> of the Benzoxaborole Pharmacophore
In this work, we present an investigation into the physical
properties
of a unique class of aromatic boronic acids, the benzoxaboroles. Using
spectrophotometric methods, the ionization constants of a family of
substituted benzoxaboroles are determined. Heterocyclic ring modifications
are examined to determine their effects on the ionization of the boronic
acid moiety. It is also shown that the substituent effects about the
aromatic ring follow a Hammett relationship with the compounds' measured
p<i>K</i><sub>a</sub> values. Finally, these substituent
effects are also shown to extend to the sugar binding properties of
these compounds under physiologically relevant conditions. Combined,
these data will inform medicinal chemists wishing to tailor the ionization
and/or ability of this class of compound to bind diol-containing biomolecules
Effects of the Donor–Acceptor Distance and Dynamics on Hydride Tunneling in the Dihydrofolate Reductase Catalyzed Reaction
A significant contemporary question in enzymology involves
the
role of protein dynamics and hydrogen tunneling in enhancing enzyme
catalyzed reactions. Here, we report a correlation between the donor–acceptor
distance (DAD) distribution and intrinsic kinetic isotope effects
(KIEs) for the dihydrofolate reductase (DHFR) catalyzed reaction.
This study compares the nature of the hydride-transfer step for a
series of active-site mutants, where the size of a side chain that
modulates the DAD (I14 in E. coli DHFR)
is systematically reduced (I14V, I14A, and I14G). The contributions
of the DAD and its dynamics to the hydride-transfer step were examined
by the temperature dependence of intrinsic KIEs, hydride-transfer
rates, activation parameters, and classical molecular dynamics (MD)
simulations. Results are interpreted within the framework of the Marcus-like
model where the increase in the temperature dependence of KIEs arises
as a direct consequence of the deviation of the DAD from its distribution
in the wild type enzyme. Classical MD simulations suggest new populations
with larger average DADs, as well as broader distributions, and a
reduction in the population of the reactive conformers correlated
with the decrease in the size of the hydrophobic residue. The more
flexible active site in the mutants required more substantial thermally
activated motions for effective H-tunneling, consistent with the hypothesis
that the role of the hydrophobic side chain of I14 is to restrict
the distribution and dynamics of the DAD and thus assist the hydride-transfer.
These studies establish relationships between the distribution of
DADs, the hydride-transfer rates, and the DAD’s rearrangement
toward tunneling-ready states. This structure–function correlation
shall assist in the interpretation of the temperature dependence of
KIEs caused by mutants far from the active site in this and other
enzymes, and may apply generally to C–H→C transfer reactions
Temporally Overlapped but Uncoupled Motions in Dihydrofolate Reductase Catalysis
Temporal correlations between protein motions and enzymatic reactions
are often interpreted as evidence for catalytically important motions.
Using dihydrofolate reductase as a model system, we show that there
are many protein motions that temporally overlapped with the chemical
reaction, and yet they do not exhibit the same kinetic behaviors (KIE
and pH dependence) as the catalyzed chemical reaction. Thus, despite
the temporal correlation, these motions are not directly coupled to
the chemical transformation, and they might represent a different
part of the catalytic cycle or simply be the product of the intrinsic
flexibility of the protein
Detection of Dihydrofolate Reductase Conformational Change by FRET Using Two Fluorescent Amino Acids
Two fluorescent amino
acids, including the novel fluorescent species
4-biphenyl-l-phenylalanine (<b>1</b>), have been incorporated
at positions 17 and 115 of dihydrofolate reductase (DHFR) to enable
a study of conformational changes associated with inhibitor binding.
Unlike most studies involving fluorescently labeled proteins, the
fluorophores were incorporated into the amino acid side chains, and
both probes [<b>1</b> and l-(7-hydroxycoumarin-4-yl)ethylglycine
(<b>2</b>)] were smaller than fluorophores typically used for
such studies. The DHFR positions were chosen as potentially useful
for Förster resonance energy transfer (FRET) measurements on
the basis of their estimated separation (17–18 Å) and
the expected change in distance along the reaction coordinate. Also
of interest was the steric accessibility of the two sites: Glu17 is
on the surface of DHFR, while Ile115 is within a folded region of
the protein. Modified DHFR I (<b>1</b> at position 17; <b>2</b> at position 115) and DHFR II (<b>2</b> at position
17; <b>1</b> at position 115) were both catalytically competent.
However, DHFR II containing the potentially rotatable biphenylphenylalanine
moiety at sterically encumbered position 115 was significantly more
active than DHFR I. Irradiation of the modified DHFRs at 280 nm effected
excitation of <b>1</b>, energy transfer to <b>2</b>, and
emission by <b>2</b> at 450 nm. However, the energy transfer
was substantially more efficient in DHFR II. The effect of inhibitor
binding was also measured. Trimethoprim mediated concentration-dependent
diminution of the emission observed at 450 nm for DHFR II but not
for DHFR I. These findings demonstrate that amino acids containing
small fluorophores can be introduced into DHFR with minimal disruption
of function and in a fashion that enables sensitive monitoring of
changes in DHFR conformation