Biology subjects

Jiricny, J.

Publications and source records attributed to Jiricny, J..

4 recordsLinked to original sources

bioRxiv2021

Prime Editing Efficiency and Fidelity are Enhanced in the Absence of Mismatch Repair

Prime editing (PE) is a powerful genome engineering approach that enables the introduction of base substitutions, insertions and deletions into any given genomic locus. However, the efficiency of PE varies widely and depends not only on the genomic region targeted, but also on the genetic background of the edited cell. To determine which cellular factors affect PE efficiency, we carried out a focused genetic screen targeting 32 DNA repair factors, spanning all reported repair pathways. We show that, depending on cell line and type of edit, ablation of mismatch repair (MMR) affords a 2-17 fold increase in PE efficiency, across several human cell lines, types of edits and genomic loci. The accumulation of the key MMR factors MLH1 and MSH2 at PE sites argues for direct involvement of MMR in PE control. Our results shed new light on the mechanism of PE and suggest how its efficiency might be optimised.

molecular biology↗

bioRxiv2020

Uncoordinated long-patch base excision repair at juxtaposed DNA lesions generates a lethal accumulation of double-strand breaks

Inhibition of the TOR pathway (TORC2, or Ypk1/2), or the depolymerization of actin filaments results in catastrophic fragmentation of the yeast genome upon exposure to low doses of the radiomimetic drug Zeocin. We find that the accumulation of double-strand breaks (DSB) is not due to altered DSB repair, but by the uncoordinated activity of base excision repair (BER) at Zeocin-modified DNA bases. We inhibit DSB formation by eliminating glycosylases and/or the endonucleases Apn1/2 and Rad1, implicating these conserved BER enzymes, or events downstream of them, in the conversion of base damage into DSBs. Among DNA polymerases, the reduction of Pol {delta}, and to a lesser extent Pol {varepsilon} and Trf4 (a Pol {beta}-like polymerase), reduces DSB formation. Finally, the BER enzymes, Ogg1 and AP endonuclease, are shown to co-precipitate with actin from yeast extracts and as purified proteins, suggesting that actin may interfere directly with the repair of Zeocin-induced damage.

molecular biology↗

bioRxiv2019

Ectopic methylation of a single persistently-unmethylated CpG in the promoter of the vitellogenin gene abolishes its inducibility by estrogen through attenuation of USF binding.

The enhancer/promoter of the vitellogenin II (VTG) gene has been extensively studied as a model system of vertebrate transcriptional control. While deletion mutagenesis and in vivo footprinting identified the transcription factor (TF) binding sites governing its tissue specificity, DNase hypersensitivity- and DNA methylation studies revealed the epigenetic changes accompanying its hormone-dependent activation. Moreover, upon induction with estrogen (E2), the region flanking the estrogen-responsive element (ERE) was reported to undergo active DNA demethylation. We now show that although the VTG ERE is methylated in embryonic chicken liver and in LMH/2A hepatocytes, its induction by E2 was not accompanied by extensive demethylation. In contrast, E2 failed to activate a VTG enhancer/promoter-controlled luciferase reporter gene methylated by SssI. Surprisingly, this inducibility difference could be traced not to the ERE, but rather to a single CpG in an E-box (CACGTG) sequence upstream of the VTG TATA box, which is unmethylated in vivo, but methylated by SssI. We demonstrate that this E-box binds the upstream stimulating factor USF1/2. Selective methylation of the CpG within this binding site with an E-box-specific DNA methyltranferase Eco72IM was sufficient to attenuate USF1/2 binding in vitro and abolish the hormone-induced transcription of the VTG gene in the reporter system.

molecular biology↗

bioRxiv2019

Transcription-coupled repair and mismatch repair contribute towards preserving genome integrity at mononucleotide repeat tracts

Introductory paragraphThe mechanisms that underpin how insertions or deletions (indels) become fixed in DNA have primarily been ascribed to replication-related and/or double-strand break (DSB)-related processes. We introduce a novel way to evaluate indels, orientating them relative to gene transcription. In so doing, we reveal a number of surprising findings: First, there is a transcriptional strand asymmetry in the distribution of mononucleotide repeat tracts in the reference human genome. Second, there is a strong transcriptional strand asymmetry of indels across 2,575 whole genome sequenced human cancers. We suggest that this is due to the activity of transcription-coupled nucleotide excision repair (TC-NER). Furthermore, TC-NER interacts with mismatch repair (MMR) under physiological conditions to produce strand bias. Finally, we show how insertions and deletions differ in their dependencies on these repair pathways. Our novel analytical approach reveals new insights into the contribution of DNA repair towards indel mutagenesis in human cells.

genomics↗