Precise mapping of single-stranded DNA breaks by using an engineered, error-prone DNA polymerase for sequence-templated erroneous end-labelling DOI Creative Commons
Ola Söderberg, Leonie Wenson, Johan Heldin

и другие.

Research Square (Research Square), Год журнала: 2024, Номер unknown

Опубликована: Окт. 7, 2024

Abstract The ability to analyze whether DNA includes lesions is important in identifying mitogenic substances. Until now, the detection of single-stranded breaks (SSBs) has lacked precise methods. To overcome this limitation, we have engineered a chimeric polymerase, Sloppymerase, that able replicate absence one nucleotide. In addition polymerase activity, Sloppymerase demonstrates 5´-3´exonuclease activity. We characterized activity and utilized enzyme develop method for sequence-templated erroneous end-labelling sequencing (STEEL-seq) relevant mapping SSBs. Following omission specific nucleotide, e.g., dATP, from reaction mixture, introduces mismatches directly downstream SSBs at positions should contain deoxyadenosine. retain sequence information after ensures hits are bona fide STEEL-seq works with variety technologies, shown by our successful experiments using Sanger, Illumina, PacBio Nanopore systems.

Язык: Английский

Protective role of extracellular vesicles against oxidative DNA damage DOI Creative Commons
Jordi Ribas‐Maynou, Ana Parra, Pablo Martínez‐Díaz

и другие.

Biological Research, Год журнала: 2025, Номер 58(1)

Опубликована: Март 13, 2025

Abstract Background Oxidative stress, a source of genotoxic damage, is often the underlying mechanism in many functional cell disorders. Extracellular vesicles (EVs) have been shown to be key regulators cellular processes and may involved maintaining redox balance. Herein, we aimed develop method assess effects EVs on DNA oxidation using porcine seminal plasma extracellular (sEVs). Results The technique was set cell-free plasmid avoid bias generated by uptake sEVs sperm cells, employing increasing concentrations hydrogen peroxide (H 2 O ) that generate single-strand breaks (SSBs). Because SSBs contain free 3’-OH end allow extension through quantitative PCR, such -and therefore SYBR intensity- showed proportional amount SSB. In next stage, H co-incubated with two size-differentiated subpopulations (small large) permeabilized non-permeabilized whether intravesicular content (IC) or surface protects from oxidative damage. obtained small reduced incidence ( P < 0.05), whereas large had no impact generation > 0.05). IC protective effect against Conclusions Our results suggest sEVs, including peripheral corona layer, exert function alterations are originated mechanisms. addition, our vitro study opens path detect, localize quantify derived different fluids, elucidating their physiopathological states.

Язык: Английский

Процитировано

0
Template-independent enzymatic functionalization of DNA oligonucleotides with environment-sensitive nucleotide probes using terminal deoxynucleotidyl transferase DOI Creative Commons
Pulak Ghosh,

Apeksha Ashok Phadte,

Bindu Bhojappa

и другие.

Nucleic Acids Research, Год журнала: 2025, Номер 53(6)

Опубликована: Март 20, 2025

Abstract Given the emerging use of terminal deoxynucleotidyl transferase (TdT) in biotechnology and its clinical potential as a cancer marker target, development versatile probe system to study processivity, substrate properties, inhibition is highly desired. Here, we demonstrate multilayered application series environment-sensitive fluorescent 2′-deoxynucleotide probes that harness activity TdT accessing site-specifically functionalized DNA oligonucleotides devising real-time fluorescence platform monitor enzyme identify inhibitors. The nucleotides constructed by coupling heterocycles progressively increasing chemical modifications (selenophene, benzothiophene, benzofuran, fluorobenzofuran) at C5 position 2′-deoxyuridine serve suitable substrates for TdT, albeit differences incorporation efficiency. A battery experiments provided valuable insights into scope this functionalization method. It revealed how fine balance between steric hindrance stacking interaction heterocycle moiety primer 3′-end nucleobase active site modulates recognition processing based on their size. Remarkably, excellent responsiveness benzofuran-modified dUTP enabled design assays estimate activity, detect nucleotide non-nucleotide findings obtained using our should significantly advance TdT-based functionalization, diagnostic, therapeutic strategies.

Язык: Английский

Процитировано

0
A massively parallelin vivoassay of TdT mutants yields variants with altered nucleotide insertion biases DOI Open Access

Courtney K. Carlson,

Theresa B. Loveless,

Marija Milisavljevic

и другие.

bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2024, Номер unknown

Опубликована: Июнь 11, 2024

Terminal deoxynucleotidyl transferase (TdT) is a unique DNA polymerase capable of template-independent extension with random nucleotides. TdT's

Язык: Английский

Процитировано

1
Bacterial Cell Lysates of Bacillus sp. F Isolated From Permafrost Diminish DNA Damage by Hydrogen Peroxide in Murine Blood Cells Ex Vivo DOI Creative Commons
Anatoli Brouchkov,

M. V. Averin,

Troy Callender

и другие.

Advanced Gut & Microbiome Research, Год журнала: 2024, Номер 2024(1)

Опубликована: Янв. 1, 2024

Nowadays, there is growing interest in new compositions and drugs that can preserve maintain human health. There are many studies on both known probiotics sources of biologically active substances which may potentially be avenues for longevity. Here, we describe the protective effect cell lysates relict bacteria Bacillus sp. F (Bacilus Lyuba), isolated from ancient permafrost Central Yakutia, upon DNA damage, induced by hydrogen peroxide. The damage was monitored a comet assay leukocytes peripheral blood experimental animals (BALB/c mice). Cell were prepared using French Press ultrasonic treatment. levels presence bacterial stress test (20 μ M peroxide 10 min at 37°C) almost twice lower, compared with buffer control. Of note, maximal observed temperature range 50°C–60°C; increase temperature, this vanished. obtained results do not give clear answer mechanism properties F. Lyuba) lysates. To question, detailed analysis protein composition lysate different temperatures should performed.

Язык: Английский

Процитировано

0
A Massively Parallel In Vivo Assay of TdT Mutants Yields Variants with Altered Nucleotide Insertion Biases DOI
Courtney Carlson, Theresa B. Loveless,

Marija Milisavljevic

и другие.

ACS Synthetic Biology, Год журнала: 2024, Номер unknown

Опубликована: Сен. 20, 2024

Terminal deoxynucleotidyl transferase (TdT) is a unique DNA polymerase capable of template-independent extension DNA. TdT's

Язык: Английский

Процитировано

0
Precise mapping of single-stranded DNA breaks by using an engineered, error-prone DNA polymerase for sequence-templated erroneous end-labelling DOI Creative Commons
Ola Söderberg, Leonie Wenson, Johan Heldin

и другие.

Research Square (Research Square), Год журнала: 2024, Номер unknown

Опубликована: Окт. 7, 2024

Abstract The ability to analyze whether DNA includes lesions is important in identifying mitogenic substances. Until now, the detection of single-stranded breaks (SSBs) has lacked precise methods. To overcome this limitation, we have engineered a chimeric polymerase, Sloppymerase, that able replicate absence one nucleotide. In addition polymerase activity, Sloppymerase demonstrates 5´-3´exonuclease activity. We characterized activity and utilized enzyme develop method for sequence-templated erroneous end-labelling sequencing (STEEL-seq) relevant mapping SSBs. Following omission specific nucleotide, e.g., dATP, from reaction mixture, introduces mismatches directly downstream SSBs at positions should contain deoxyadenosine. retain sequence information after ensures hits are bona fide STEEL-seq works with variety technologies, shown by our successful experiments using Sanger, Illumina, PacBio Nanopore systems.

Язык: Английский

Процитировано

0