Networks of redox sensor proteins within discrete microdomains regulate the flow of redox signaling. Yet, the inherent reactivity of redox signals complicates the study of specific redox events and pathways by traditional methods. Herein, we review designer chemistries capable of measuring flux and/or mimicking subcellular redox signaling at the cellular and organismal level. Such efforts have begun to decipher the logic underlying organelle-, site-, and target-specific redox signaling in vitro and in vivo. These data highlight chemical biology as a perfect gateway to interrogate how Nature choreographs subcellular redox chemistry to drive precision redox biology.
DNA interstrand cross-links are an
important family of DNA damage
that block replication and transcription. Recently, it was discovered
that oxidized abasic sites react with the opposing strand of DNA to
produce interstrand cross-links. Some of the cross-links between 2′-deoxyadenosine
and the oxidized abasic sites, 5′-(2-phosphoryl-1,4-dioxobutane)
(DOB) and the C4-hydroxylated abasic site (C4-AP), are formed reversibly.
Chemical instability hinders biochemical, structural, and physicochemical
characterization of these cross-linked duplexes. To overcome these
limitations, we developed methods for preparing stabilized analogues
of DOB and C4-AP cross-links via solid-phase oligonucleotide synthesis.
Oligonucleotides of any sequence are attainable by synthesizing phosphoramidites
in which the hydroxyl groups of the cross-linked product were orthogonally
protected using photochemically labile and hydrazine labile groups.
Selective unmasking of a single hydroxyl group precedes solid-phase
synthesis of one arm of the cross-linked DNA. The method is compatible
with commercially available phosphoramidites and other oligonucleotide
synthesis reagents. Cross-linked duplexes containing as many as 54
nt were synthesized on solid-phase supports. Subsequent enzyme ligation
of one cross-link product provided a 60 bp duplex, which is suitable
for nucleotide excision repair studies.
5′-(2-Phosphoryl-1,4-dioxobutane) (DOB) is an oxidized abasic site that is produced by several antitumor agents and γ-radiolysis. DOB reacts reversibly with a dA opposite the 3′-adjacent nucleotide to form DNA interstrand cross-links (ICLs), genotoxic DNA lesions that can block DNA replication and transcription. Translesion synthesis (TLS) is an important step in several ICL repair pathways to bypass unhooked intermediates generated by endonucleolytic incision. The instability of DOB-ICLs has made it difficult to learn about their TLS-mediated repair capability and mutagenic potential. We recently developed a method for chemically synthesizing oligonucleotides containing a modified DOB-ICL analogue. Herein, we examined the capabilities of several highly relevant eukaryotic TLS DNA polymerases (pols), including human pol η, pol κ, pol ι, pol ν, REV1, and yeast pol ζ, to bypass this DOB-ICL analogue. The prelesion, translesion, and postlesion replication efficiency and fidelity were examined. Pol η showed moderate bypass activity when encountering the DOB-ICL, giving major products one or two nucleotides beyond the cross-linked template nucleotide. In contrast, DNA synthesis by the other pols was stalled at the position before the cross-linked nucleotide. Steady-state kinetic data and liquid chromatography–mass spectrometry sequencing of primer extension products by pol η unambiguously revealed that pol η-mediated bypass is highly error-prone. Together, our study provides the first set of in vitro evidence that the DOB-ICL is a replication-blocking and highly miscoding lesion. Compared to several other TLS pols examined, pol η is likely to contribute to the TLS-mediated repair of the DOB-ICL in vivo.
Nucleotide
excision repair is a primary pathway in cells for coping
with DNA interstrand cross-links (ICLs). Recently, C4′-oxidized
(C4-AP) and C5′-oxidized abasic sites (DOB) that are produced
following hydrogen atom abstraction from the DNA backbone were found
to produce ICLs. Because some of the ICLs derived from C4-AP and DOB
are too unstable to characterize in biochemical processes, chemically
stable analogues were synthesized [Ghosh, S., and Greenberg, M. M.
(2014) J. Org. Chem.79, 5948–5957].
UvrABC incision of DNA substrates containing stabilized analogues
of the ICLs derived from C4-AP and DOB was examined. The incision
pattern for the ICL related to the C4′-oxidized abasic site
was typical for UvrABC substrates. UvrABC cleaved both strands of
the substrate containing the C4-AP ICL analogue, but it was a poor
substrate. UvrABC incised <30% of the C4-AP ICL analogue over an
8 h period, raising the possibility that this cross-link will be inefficiently
repaired in cells. Furthermore, double-strand breaks were not detected
upon incision of an internally labeled hairpin substrate containing
the C4-AP ICL analogue. UvrABC incised the stabilized analogue of
the DOB ICL more efficiently (∼20% in 1 h). Furthermore, the
incision pattern was unique, and the cross-linked substrate was converted
into a single product, a double-strand break. The template strand
was exclusively incised on the template strand on the 3′-side
of the cross-linked dA. Although the outcomes of the interaction between
UvrABC and these two cross-linked substrates are different from one
another, they provide additional examples of how seemingly simple
lesions (C4-AP and DOB) can potentially exert significant deleterious
effects on biochemical processes.
We present methods for limited-resource-friendly preparation of chitosan magnetic particles that are amenable to ultrasensitive downstream isothermal and conventional nucleic acid amplification tests.
Fabricated an inexpensive bi-electrode electrochemical sensor for nucleic acid amplification detection with a user-friendly interface in a short detection time.Electronic conduction of the metal oxide sensing layer was successfully optimised by manipulating the concentration of oxygen vacancies.The sensing layer was annealed at various temperatures and optimised by testing their electrical, optical, and chemical properties.The fabricated device was able to detect dengue virus sequence DNA and Staphylococcus aureus genomic DNA with clinically relevant limit of detection.
C4′-oxidized (C4-AP) and C5′-oxidized abasic sites (DOB) that are produced following abstraction of a hydrogen atom from the DNA backbone reversibly form cross-links selectively with dA opposite a 3′-adjacent nucleotide, despite the comparable proximity of an opposing dA. A previous report on UvrABC incision of DNA substrates containing stabilized analogues of the ICLs derived from C4-AP and DOB also indicated that the latter is repaired more readily by nucleotide excision repair [Ghosh, S., and Greenberg, M. M. (2014) Biochemistry 53, 5958–5965]. The source for selective cross-link formation was probed by comparing the reactivity of ICL analogues of C4-AP and DOB that mimic the preferred and disfavored cross-links with that of reagents that indirectly detect distortion by reacting with the nucleobases. The disfavored C4-AP and DOB analogues were each more reactive than the corresponding preferred cross-link substrates, suggesting that the latter are more stable, which is consistent with selective ICL formation. In addition, the preferred DOB analogue is more reactive than the respective C4-AP ICL, which is consistent with its more efficient incision by UvrABC. The conclusions drawn from the chemical probing experiments are corroborated by UV melting studies. The preferred ICLs exhibit melting temperatures higher than those of the corresponding disfavored isomers. These studies suggest that oxidized abasic sites form reversible interstrand cross-links with dA opposite the 3′-adjacent thymidine because these products are more stable and the thermodynamic preference is reflected in the transition states for their formation.
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