5-(Hydroxymethyl)uracil and -cytosine as potential epigenetic marks enhancing or inhibiting transcription with bacterial RNA polymerase

Janoušková, Martina; Vaníková, Zuzana; Nici, Fabrizia; Boháčová, Soňa; Vítovská, Dragana; Šanderová, Hana; Hocek, Michal; Krásný, Libor

doi:10.1039/c7cc08053k

Cited by 20 publications

(41 citation statements)

References 32 publications

(22 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Next, we tested dU hm TP, dU NB TP, dU NP TP and dU An TP in PCR using either a 98-mer or (a more demanding) 297-mer template. In agreement with our previous communications, 17,22 the PCR reactions with dU hm TP and dU NB TP, as well as with the new nitropiperonyl-caged dU NP TP, proceeded but the products of amplification were rather weak. The agarose gel of the 98-mer PCR (Fig.…”

Section: Incorporation Of Caged Nucleotides Into Dnasupporting

confidence: 92%

See 1 more Smart Citation

Protected 5-(hydroxymethyl)uracil nucleotides bearing visible-light photocleavable groups as building blocks for polymerase synthesis of photocaged DNA

et al. 2018

Self Cite

View full text Add to dashboard Cite

Nucleosides, nucleotides and 2'-deoxyribonucleoside triphosphates (dNTPs) containing 5-(hydroxymethyl)uracil protected with photocleavable groups (2-nitrobenzyl-, 6-nitropiperonyl or 9-anthrylmethyl) were prepared and tested as building blocks for the polymerase synthesis of photocaged oligonucleotides and DNA. Photodeprotection (photorelease) reactions were studied in detail on model nucleoside monophosphates and their photoreaction quantum yields were determined. Photocaged dNTPs were then tested and used as substrates for DNA polymerases in primer extension or PCR. DNA probes containing photocaged or free 5-hydroxymethylU in the recognition sequence of restriction endonucleases were prepared and used for the study of photorelease of caged DNA by UV or visible light at different wavelengths. The nitropiperonyl-protected nucleotide was found to be a superior building block because the corresponding dNTP is a good substrate for DNA polymerases, and the protecting group is efficiently cleavable by irradiation by UV or visible light (up to 425 nm).

show abstract

Section: Incorporation Of Caged Nucleotides Into Dnasupporting

confidence: 92%

“…Obviously, any bulkier modifications in the major groove inhibited the transcription. 16 Recently we found 17 that the presence of 5hmU or 5hmC in template DNA can even enhance (up to 3.5 times) or inhibit bacterial transcription (depending on the promoter) and thus even hmU could be an epigenetic mark.…”

Section: Introductionmentioning

confidence: 99%

Protected 5-(hydroxymethyl)uracil nucleotides bearing visible-light photocleavable groups as building blocks for polymerase synthesis of photocaged DNA

et al. 2018

Self Cite

View full text Add to dashboard Cite

show abstract

“…Introduction 5-(Hydroxymethyl)cytosine (5hmC) is an epigenetic DNA base 1 which is not only an intermediate in active DNA demethylation 2 but also an epigenetic signal regulating gene expression. 3,4 Several protected 5hmC 2′-deoxyribonucleoside building blocks for the phosphoramidite synthesis of modified oligonucleotides (ONs) have been reported, 5 as well as the synthesis and use of the corresponding unprotected 5hmC 2′deoxyribonucleoside triphosphate (dC hm TP) in the polymerase synthesis of modified DNA. 6 For a deeper study of the role of 5hmC in the regulation of transcription, 3,4 genomic stability and active demethylation, 2 it might be advantageous to have a masked/caged 5hmC, which can be released in DNA on demand.…”

mentioning

confidence: 99%

Protected 2′-deoxyribonucleoside triphosphate building blocks for the photocaging of epigenetic 5-(hydroxymethyl)cytosine in DNA

et al. 2018

Self Cite

View full text Add to dashboard Cite

2'-Deoxyribonucleoside triphosphates (dNTPs) containing 5-(hydroxymethyl)cytosine (5hmC) protected with photocleavable groups (2-nitrobenzyl or 6-nitropiperonyl) were prepared and studied as substrates for the enzymatic synthesis of oligonucleotides and DNA containing a photocaged epigenetic 5hmC base. DNA probes containing photocaged or free 5hmC in the recognition sequence of restriction endonucleases were prepared and used for the study of the photorelease of caged DNA by UV or visible light at different wavelengths. The nitrobenzyl-protected dNTP was a slightly better substrate for DNA polymerases in primer extension or PCR, whereas the nitropiperonyl-protected nucleotide underwent slightly faster photorelease at 400 nm. However, both photocaged building blocks can be used in polymerase synthesis and the photorelease of 5hmC in DNA.

show abstract

“…[6] In mammals,reported levels of 5hmU vary by celland tissue types. [2b, 7] Increased levels of 5hmU autoantibodies have been reported in cancer cases, [8] and blood 5hmU mononucleoside levels have been studied as am arker of cancer risks and invasiveness.…”

Section: Dna-base Modifications Can Profoundly Influence Biologymentioning

confidence: 99%

“…[5] When incorporated at some promoter sites, 5hmU has been shown to affect transcription by bacterial RNAP,therefore it may have asignificant effect on microbial biology. [6] In mammals,reported levels of 5hmU vary by celland tissue types. [2b, 7] Increased levels of 5hmU autoantibodies have been reported in cancer cases, [8] and blood 5hmU mononucleoside levels have been studied as am arker of cancer risks and invasiveness.…”

Section: Dna-base Modifications Can Profoundly Influence Biologymentioning

confidence: 99%

Sequencing 5‐Hydroxymethyluracil at Single‐Base Resolution

et al. 2018

View full text Add to dashboard Cite

5-hydroxymethyluracil (5hmU) is formed through oxidation of thymine both enzymatically and non-enzymatically in various biological systems.A lthough 5hmU has been reported to affect biological processes such as protein-DNA interactions,the consequences of 5hmU formation in genomes have not been yet fully explored. Herein, we report amethod to sequence 5hmU at single-base resolution. We employ chemical oxidation to transform 5hmU to 5-formyluracil (5fU), followed by the polymerase extension to induce T-to-C base changes owingt ot he inherent ability of 5fUt of orm 5fU:G base pairing. In combination with the Illumina next generation sequencing technology,w ed eveloped polymerase chain reaction (PCR) conditions to amplify the T-to-C base changes and demonstrate the method in three different synthetic oligonucleotide models as well as part of the genome of a5 hmU-rich eukaryotic pathogen. Our method has the potential capability to map 5hmU in genomic DNAa nd thus will contribute to promote the understanding of this modified base. DNA-base modifications can profoundly influence biologyand an umber of modified bases have been identified in the genomes of av ariety of organisms.[1] 5-hydroxymethyluracil (5hmU) is produced through oxidation of thymine both enzymatically and non-enzymatically [2] and can influence the binding of proteins to DNA. [2b,3] It has also been suggested that 5hmU might lead to genomic instability as it can be removed by DNAr epair enzymes to create potentially mutagenic lesions [4] and affect the stability of DNA duplexes.[5] When incorporated at some promoter sites, 5hmU has been shown to affect transcription by bacterial RNAP,therefore it may have asignificant effect on microbial biology. [6] In mammals,reported levels of 5hmU vary by celland tissue types. [2b, 7] Increased levels of 5hmU autoantibodies have been reported in cancer cases, [8] and blood 5hmU mononucleoside levels have been studied as am arker of cancer risks and invasiveness.[9] We previously reported am ethod to map 5hmU at moderate resolution by chemical enrichment of 5hmU-containing DNAfragments followed by sequencing.[10] Amethod for single-base sequencing of 5hmU would enable the identification of individual modification sites in genomes.Asingle-molecule real-time (SMRT) sequencing approach could in principle be applied to map 5hmU at single-base resolution, however the intrinsic signature signal for 5hmU is rather weak unless the base is further modified.[11] Mapping 5hmU at single-base resolution is aw orthy challenge that could transform genome-wide analysis of 5hmU.H erein, we describe ac hemical approach for single base-resolution sequencing of 5hmU and demonstrate its utility in various sequence contexts.Thec onceptual basis for sequencing 5hmU involves chemical oxidation of 5hmU to 5fU, which ionizes under mild alkaline pH owing to the electron-withdrawing exocyclic aldehyde (pK a at N3 = 8.1 for 5fUv s. 9.3 for 5hmU).[12] The ionized form of 5fUcan base-pair with G (Figure 1), causing aT -to-C base chan...

show abstract

5-(Hydroxymethyl)uracil and -cytosine as potential epigenetic marks enhancing or inhibiting transcription with bacterial RNA polymerase

Cited by 20 publications

References 32 publications

Protected 5-(hydroxymethyl)uracil nucleotides bearing visible-light photocleavable groups as building blocks for polymerase synthesis of photocaged DNA

Protected 5-(hydroxymethyl)uracil nucleotides bearing visible-light photocleavable groups as building blocks for polymerase synthesis of photocaged DNA

Protected 2′-deoxyribonucleoside triphosphate building blocks for the photocaging of epigenetic 5-(hydroxymethyl)cytosine in DNA

Sequencing 5‐Hydroxymethyluracil at Single‐Base Resolution

Contact Info

Product

Resources

About