6 research outputs found
The Miscoding Potential of 5-Hydroxycytosine Arises Due to Template Instability in the Replicative Polymerase Active Site
5-Hydroxycytosine (5-OHC) is a stable oxidation product
of cytosine associated with an increased frequency of C → T
transition mutations. When this lesion escapes recognition by the
base excision repair pathway and persists to serve as a templating
base during DNA synthesis, replicative DNA polymerases often misincorporate
dAMP at the primer terminus,
which can lead to fixation of mutations
and subsequent disease. To characterize the dynamics of DNA synthesis
opposite 5-OHC, we initiated a comparison of unmodified dCMP to 5-OHC,
5-fluorocytosine (5-FC), and 5-methylcytosine (5-MEC) in which these
bases act as templates in the active site of RB69 gp43, a high-fidelity
DNA polymerase sharing homology with human replicative DNA polymerases.
This study presents the first crystal structure of any DNA polymerase
binding this physiologically important premutagenic DNA lesion, showing
that while dGMP is stabilized by 5-OHC through normal Watson–Crick
base pairing, incorporation of dAMP leads to unstacking and instability
in the template. Furthermore, the electronegativity of the C5 substituent
appears to be important in the miscoding potential of these cytosine-like
templates. While dAMP is incorporated opposite 5-OHC ∼5 times
more efficiently than opposite unmodified dCMP, an elevated level
of incorporation is also observed opposite 5-FC but not 5-MEC. Taken
together, these data imply that the nonuniform templating by 5-OHC
is due to weakened stacking capabilities, which allows dAMP incorporation
to proceed in a manner similar to that observed opposite abasic sites
The A‑Rule and Deletion Formation During Abasic and Oxidized Abasic Site Bypass by DNA Polymerase θ
DNA polymerase θ (Pol θ)
is implicated in various cellular
processes including double-strand break repair and apurinic/apyrimidinic
site bypass. Because Pol θ expression correlates with poor cancer
prognosis, the ability of Pol θ to bypass the C4′-oxidized
abasic site (C4-AP) and 2-deoxyribonolactone (L), which are generated
by cytotoxic agents, is of interest. Translesion synthesis and subsequent
extension by Pol θ past C4-AP or L and an abasic site (AP) or
its tetrahydrofuran analogue (F) was examined. Pol θ conducts
translesion synthesis on templates containing AP and F with similar
efficiencies and follows the “A-rule,” inserting nucleotides
in the order A > G > T. Translesion synthesis on templates containing
C4-AP and L is less efficient than AP and F, and the preference for
A insertion is reduced for L and absent for C4-AP. Extension past
all abasic lesions (AP, F, C4-AP, and L) was significantly less efficient
than translesion synthesis and yielded deletions caused by the base
one or two nucleotides downstream from the lesion being used as a
template, with the latter being favored. These results suggest that
bypass of abasic lesions by Pol θ is highly mutagenic
Crystal Structure of DNA Polymerase β with DNA Containing the Base Lesion Spiroiminodihydantoin in a Templating Position
The
first high-resolution crystal structure of spiroiminodihydantoin
(dSp1) was obtained in the context of the DNA polymerase β active
site and reveals two areas of significance. First, the structure verifies
the recently determined <i>S</i> configuration at the spirocyclic
carbon. Second, the distortion of the DNA duplex is similar to that
of the single-oxidation product 8-oxoguanine. For both oxidized lesions,
adaptation of the <i>syn</i> conformation results in similar
backbone distortions in the DNA duplex. The resulting conformation
positions the dSp1 A-ring as the base-pairing face whereas the B-ring
of dSp1 protrudes into the major groove
Remote Mutations Induce Functional Changes in Active Site Residues of Human DNA Polymerase β
With the formidable
growth in the volume of genetic information,
it has become essential to identify and characterize mutations in
macromolecules not only to predict contributions to disease processes
but also to guide the design of therapeutic strategies. While mutations
of certain residues have a predictable phenotype based on their chemical
nature and known structural position, many types of mutations evade
prediction based on current information. Described in this work are
the crystal structures of two cancer variants located in the palm
domain of DNA polymerase β (pol β), S229L and G231D, whose
biological phenotype was not readily linked to a predictable structural
implication. Structural results demonstrate that the mutations elicit
their effect through subtle influences on secondary interactions with
a residue neighboring the active site. Residues 229 and 231 are 7.5
and 12.5 Å, respectively, from the nearest active site residue,
with a β-strand between them. A residue on this intervening
strand, M236, appears to transmit fine structural perturbations to
the catalytic metal-coordinating residue D256, affecting its conformational
stability
Defective Nucleotide Release by DNA Polymerase β Mutator Variant E288K Is the Basis of Its Low Fidelity
DNA
polymerases synthesize new DNA during DNA replication and repair,
and their ability to do so faithfully is essential to maintaining
genomic integrity. DNA polymerase β (Pol β) functions
in base excision repair to fill in single-nucleotide gaps, and variants
of Pol β have been associated with cancer. Specifically, the
E288K Pol β variant has been found in colon tumors and has been
shown to display sequence-specific mutator activity. To probe the
mechanism that may underlie E288K’s loss of fidelity, a fluorescence
resonance energy transfer system that utilizes a fluorophore on the
fingers domain of Pol β and a quencher on the DNA substrate
was employed. Our results show that E288K utilizes an overall mechanism
similar to that of wild type (WT) Pol β when incorporating correct
dNTP. However, when inserting the correct dNTP, E288K exhibits a faster
rate of closing of the fingers domain combined with a slower rate
of nucleotide release compared to those of WT Pol β. We also
detect enzyme closure upon mixing with the incorrect dNTP for E288K
but not WT Pol β. Taken together, our results suggest that E288K
Pol β incorporates all dNTPs more readily than WT because of
an inherent defect that results in rapid isomerization of dNTPs within
its active site. Structural modeling implies that this inherent defect
is due to interaction of E288K with DNA, resulting in a stable closed
enzyme structure
Defective Nucleotide Release by DNA Polymerase β Mutator Variant E288K Is the Basis of Its Low Fidelity
DNA
polymerases synthesize new DNA during DNA replication and repair,
and their ability to do so faithfully is essential to maintaining
genomic integrity. DNA polymerase β (Pol β) functions
in base excision repair to fill in single-nucleotide gaps, and variants
of Pol β have been associated with cancer. Specifically, the
E288K Pol β variant has been found in colon tumors and has been
shown to display sequence-specific mutator activity. To probe the
mechanism that may underlie E288K’s loss of fidelity, a fluorescence
resonance energy transfer system that utilizes a fluorophore on the
fingers domain of Pol β and a quencher on the DNA substrate
was employed. Our results show that E288K utilizes an overall mechanism
similar to that of wild type (WT) Pol β when incorporating correct
dNTP. However, when inserting the correct dNTP, E288K exhibits a faster
rate of closing of the fingers domain combined with a slower rate
of nucleotide release compared to those of WT Pol β. We also
detect enzyme closure upon mixing with the incorrect dNTP for E288K
but not WT Pol β. Taken together, our results suggest that E288K
Pol β incorporates all dNTPs more readily than WT because of
an inherent defect that results in rapid isomerization of dNTPs within
its active site. Structural modeling implies that this inherent defect
is due to interaction of E288K with DNA, resulting in a stable closed
enzyme structure