Many DNA-processing enzymes have been shown to contain a 4Fe4S cluster, a common redox cofactor in biology. Using DNA electrochemistry, we find that binding of the DNA polyanion promotes a negative ...shift in 4Fe4S cluster potential, which corresponds thermodynamically to a ∼500-fold increase in DNA-binding affinity for the oxidized 4Fe4S
3+
cluster versus the reduced 4Fe4S
2+
cluster. This redox switch can be activated from a distance using DNA charge transport (DNA CT) chemistry. DNA-processing proteins containing the 4Fe4S cluster are enumerated, with possible roles for the redox switch highlighted. A model is described where repair proteins may signal one another using DNA-mediated charge transport as a first step in their search for lesions. The redox switch in eukaryotic DNA primases appears to regulate polymerase handoff, and in DNA polymerase δ, the redox switch provides a means to modulate replication in response to oxidative stress. We thus describe redox signaling interactions of DNA-processing 4Fe4S enzymes, as well as the most interesting potential players to consider in delineating new DNA-mediated redox signaling networks.
UvrC Coordinates an O2‑Sensitive [4Fe4S] Cofactor Silva, Rebekah M. B; Grodick, Michael A; Barton, Jacqueline K
Journal of the American Chemical Society,
06/2020, Volume:
142, Issue:
25
Journal Article
Peer reviewed
Open access
Recent advances have led to numerous landmark discoveries of 4Fe4S clusters coordinated by essential enzymes in repair, replication, and transcription across all domains of life. The cofactor has ...notably been challenging to observe for many nucleic acid processing enzymes due to several factors, including a weak bioinformatic signature of the coordinating cysteines and lability of the metal cofactor. To overcome these challenges, we have used sequence alignments, an anaerobic purification method, iron quantification, and UV–visible and electron paramagnetic resonance spectroscopies to investigate UvrC, the dual-incision endonuclease in the bacterial nucleotide excision repair (NER) pathway. The characteristics of UvrC are consistent with 4Fe4S coordination with 60–70% cofactor incorporation, and additionally, we show that, bound to UvrC, the 4Fe4S cofactor is susceptible to oxidative degradation with aggregation of apo species. Importantly, in its holo form with the cofactor bound, UvrC forms high affinity complexes with duplexed DNA substrates; the apparent dissociation constants to well-matched and damaged duplex substrates are 100 ± 20 nM and 80 ± 30 nM, respectively. This high affinity DNA binding contrasts reports made for isolated protein lacking the cofactor. Moreover, using DNA electrochemistry, we find that the cluster coordinated by UvrC is redox-active and participates in DNA-mediated charge transport chemistry with a DNA-bound midpoint potential of 90 mV vs NHE. This work highlights that the 4Fe4S center is critical to UvrC.
Recent advances have led to numerous landmark discoveries of 4Fe4S clusters coordinated by essential enzymes in repair, replication, and transcription across all domains of life. The cofactor has ...notably been challenging to observe for many nucleic acid processing enzymes due to several factors, including a weak bioinformatic signature of the coordinating cysteines and lability of the metal cofactor. To overcome these challenges, we have used sequence alignments, an anaerobic purification method, iron quantification, and UV-visible and electron paramagnetic resonance spectroscopies to investigate UvrC, the dual-incision endonuclease in the bacterial nucleotide excision repair (NER) pathway. The characteristics of UvrC are consistent with 4Fe4S coordination with 60-70% cofactor incorporation, and additionally, we show that, bound to UvrC, the 4Fe4S cofactor is susceptible to oxidative degradation with aggregation of apo species. Importantly, in its holo form with the cofactor bound, UvrC forms high affinity complexes with duplexed DNA substrates; the apparent dissociation constants to well-matched and damaged duplex substrates are 100 ± 20 nM and 80 ± 30 nM, respectively. This high affinity DNA binding contrasts reports made for isolated protein lacking the cofactor. Moreover, using DNA electrochemistry, we find that the cluster coordinated by UvrC is redox-active and participates in DNA-mediated charge transport chemistry with a DNA-bound midpoint potential of 90 mV vs NHE. This work highlights that the 4Fe4S center is critical to UvrC.
Redox Signaling through DNA O'Brien, Elizabeth; Silva, Rebekah M. B.; Barton, Jacqueline K.
Israel journal of chemistry,
October 2016, Volume:
56, Issue:
9-10
Journal Article
Peer reviewed
Open access
Biological electron transfer reactions between metal cofactors are critical to many essential processes within the cell. Duplex DNA is, moreover, capable of mediating the transport of charge through ...its π‐stacked nitrogenous bases. Increasingly, 4Fe4S clusters, generally redox‐active cofactors, have been found to be associated with enzymes involved in DNA processing. DNA‐binding enzymes containing 4Fe4S clusters can thus utilize DNA charge transport (DNA CT) for redox signaling to coordinate reactions over long molecular distances. In particular, DNA CT signaling may represent the first step in the search for DNA lesions by proteins containing 4Fe4S clusters that are involved in DNA repair. Here we describe research carried out to examine the chemical characteristics and biological consequences of DNA CT. We are finding that DNA CT among metalloproteins represents powerful chemistry for redox signaling at long range within the cell.
Many DNA-processing enzymes have been shown to contain a 4Fe4S cluster, a common redox cofactor in biology. We find using DNA electrochemistry that binding of the DNA polyanion promotes a negative ...shift in 4Fe4S cluster potential, which corresponds thermodynamically to ~ 500-fold increase in DNA binding affinity for the oxidized 4Fe4S
3+
cluster versus the reduced 4Fe4S
2+
cluster. This redox switch can be activated from a distance using DNA charge transport chemistry. DNA-processing proteins containing the 4Fe4S cluster are enumerated with possible roles for the redox switch, highlighted. A model is described where repair proteins may signal one another using DNA-mediated charge transport as a first step in their search for lesions. The redox switch in eukaryotic DNA primases appears to regulate polymerase handoff, and in DNA polymerase δ, the redox switch provides a means to modulate replication in response to oxidative stress. Thus we describe redox signaling interactions of DNA-processing 4Fe4S enzymes as well as the most interesting potential players to consider in delineating new DNA-mediated redox signaling networks.
Understanding SARS-CoV-2 transmission in higher education settings is important to limit spread between students, and into at-risk populations. In this study, we sequenced 482 SARS-CoV-2 isolates ...from the University of Cambridge from 5 October to 6 December 2020. We perform a detailed phylogenetic comparison with 972 isolates from the surrounding community, complemented with epidemiological and contact tracing data, to determine transmission dynamics. We observe limited viral introductions into the university; the majority of student cases were linked to a single genetic cluster, likely following social gatherings at a venue outside the university. We identify considerable onward transmission associated with student accommodation and courses; this was effectively contained using local infection control measures and following a national lockdown. Transmission clusters were largely segregated within the university or the community. Our study highlights key determinants of SARS-CoV-2 transmission and effective interventions in a higher education setting that will inform public health policy during pandemics.
Mitigation of SARS-CoV-2 transmission from international travel is a priority. We evaluated the effectiveness of travellers being required to quarantine for 14-days on return to England in Summer ...2020. We identified 4,207 travel-related SARS-CoV-2 cases and their contacts, and identified 827 associated SARS-CoV-2 genomes. Overall, quarantine was associated with a lower rate of contacts, and the impact of quarantine was greatest in the 16-20 age-group. 186 SARS-CoV-2 genomes were sufficiently unique to identify travel-related clusters. Fewer genomically-linked cases were observed for index cases who returned from countries with quarantine requirement compared to countries with no quarantine requirement. This difference was explained by fewer importation events per identified genome for these cases, as opposed to fewer onward contacts per case. Overall, our study demonstrates that a 14-day quarantine period reduces, but does not completely eliminate, the onward transmission of imported cases, mainly by dissuading travel to countries with a quarantine requirement.
Pellagra is caused by niacin (vitamin B3) deficiency and patients with pellagra present with a characteristic rash. Isoniazid disrupts intracellular niacin synthesis and might induce niacin ...deficiency. In 2017, Malawi scaled up continuous isoniazid preventive treatment (IPT) for tuberculosis prevention among people living with HIV. In addition, an under-diversified diet based on subsistence maize, as is commonly the case in Malawi, is a risk factor for pellagra. We aimed to investigate whether large-scale isoniazid exposure in Malawi contributed to the cumulative risk for pellagra in a nutritionally vulnerable population.
We did a matched case-control study to evaluate the association between daily, continuous isoniazid exposure and pellagra. We matched sequentially enrolled patients with pellagra each with four control participants by sex and age from referral dermatology centres in three IPT scale-up districts in Malawi (Lilongwe, Blantyre, and Zomba) to evaluate isoniazid as a risk for pellagra using multivariable conditional logistic regression. We established a community clinic referral system surrounding the dermatology clinic in each district to enhance case-finding and included all patients with pellagra, regardless of referral status. The primary outcome was dermatologist-diagnosed pellagra. We calculated the interval between isoniazid initiation and rash onset and assessed 30-day clinical outcomes after multi-B vitamin treatment containing 300 mg nicotinamide daily.
Between Feb 5 and Aug 9, 2019, we enrolled 197 patients with pellagra and 781 matched controls. Isoniazid exposure was associated with an increased risk of pellagra (adjusted odds ratio 42·6 95% CI 13·3–136·6). Significant covariates included HIV infection, referral status, food insecurity, underweight, excess alcohol consumption, and, among women, lactation. The median time from isoniazid initiation to rash onset was shorter during the season of food scarcity (5 months IQR 3–7) compared with the harvest season (9 months 8–11; hazard ratio 7·2 95% CI 3·2–16·2, log-rank p<0·0001). Those with isoniazid-associated pellagra who discontinued isoniazid and adhered to multi-B vitamin treatment showed 30-day clinical improvement.
Continuous IPT scale-up and the annual period of food scarcity both increased the risk of pellagra in Malawi. Use of shorter rifamycin-based regimens for tuberculosis prevention and food fortification in populations with undernutrition might reduce this risk. Niacin-containing multi-B vitamin co-administration with isoniazid as pellagra prevention is worth exploring further.
This study was supported by the President's Emergency Plan for AIDS Relief through the US Centers for Disease Control and Prevention under project 7173.
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