Targeted DNA ADP-ribosylation triggers templated repair in bacteria and base mutagenesis in eukaryotes

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

Опубликована: Ноя. 17, 2024

Base editors create precise genomic edits by directing nucleobase deamination or removal without inducing double-stranded DNA breaks. However, a vast chemical space of other modifications remains to be explored for genome editing. Here, we harness the bacterial anti-phage toxin DarT2 append ADP-ribosyl moieties DNA, unlocking distinct editing outcomes in bacteria versus eukaryotes. Fusing an attenuated Cas9 nickase, program site-specific ADP-ribosylation thymines within target sequence. In tested bacteria, targeting drives efficient homologous recombination offering flexible and scar-free base replacement nor counterselection. eukaryotes including yeast, plants human cells, substitution modified thymine adenine mixture cytosine with limited insertions deletions, inaccessible current editors. Altogether, our approach, called editing, leverages addition moiety expand modalities precision gene

Язык: Английский

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REV1 Coordinates a Multi-Faceted Tolerance Response to DNA Alkylation Damage and Prevents Chromosome Shattering inDrosophila melanogaster

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

Опубликована: Фев. 13, 2024

Abstract When replication forks encounter damaged DNA, cells utilize DNA damage tolerance mechanisms to allow proceed. These include translesion synthesis at the fork, postreplication gap filling, and template switching via fork reversal or homologous recombination. The extent which these different are utilized depends on cell, tissue, developmental context-specific cues, last two of poorly understood. To address this gap, we have investigated responses following alkylation in Drosophila melanogaster . We report that synthesis, rather than switching, is preferred response alkylation-induced diploid larval tissues. Furthermore, show REV1 protein plays a multi-faceted role Drosophila. larvae lacking hypersensitive methyl methanesulfonate (MMS) highly elevated levels γ-H2Av foci chromosome aberrations MMS-treated Loss C-terminal domain (CTD), recruits multiple polymerases sites, sensitizes flies MMS. In absence CTD, eta zeta become critical for MMS tolerance. addition, REV3, catalytic subunit polymerase zeta, require deoxycytidyl transferase activity tolerate Together, our results demonstrate prioritize use highlight coordination prevent genome instability. Author Summary Organisms evolved several ways continue copying their when it damaged, grouped into categories switching. from collapsing they catastrophic instability cell death. While proteins pathways involved beginning be understood single level, how regulated multicellular organisms an intriguing question. study, by during development. discovered tissues containing rapidly dividing favor over preferentially utilizing context-dependent manner. showed protein, best known its recruiting performs functions preferences may differ those observed cultured cells, establish as useful model system studying mechanisms.

Язык: Английский

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REV1 coordinates a multi-faceted tolerance response to DNA alkylation damage and prevents chromosome shattering in Drosophila melanogaster

PLoS Genetics, Год журнала: 2024, Номер 20(7), С. e1011181 - e1011181

Опубликована: Июль 29, 2024

When replication forks encounter damaged DNA, cells utilize damage tolerance mechanisms to allow proceed. These include translesion synthesis at the fork, postreplication gap filling, and template switching via fork reversal or homologous recombination. The extent which these different are utilized depends on cell, tissue, developmental context-specific cues, last two of poorly understood. To address this gap, we have investigated responses in Drosophila melanogaster. We report that DNA alkylation rapidly dividing larval tissues heavily synthesis. Furthermore, show REV1 protein plays a multi-faceted role Drosophila. Larvae lacking hypersensitive methyl methanesulfonate (MMS) highly elevated levels γ-H2Av (Drosophila γ-H2AX) foci chromosome aberrations MMS-treated tissues. Loss C-terminal domain (CTD), recruits multiple polymerases sites, sensitizes flies MMS. In absence CTD, eta zeta become critical for MMS tolerance. addition, REV3, catalytic subunit polymerase zeta, require deoxycytidyl transferase activity tolerate Together, our results demonstrate prioritize use highlight coordination response prevent genome instability.

Язык: Английский

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Molecular identification of a peroxidase gene controlling body size in the entomopathogenic nematodeSteinernema hermaphroditum

bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2023, Номер unknown

Опубликована: Ноя. 10, 2023

Abstract The entomopathogenic nematode Steinernema hermaphroditum was recently rediscovered and is being developed as a genetically tractable experimental system for the study of previously unexplored biology, including parasitism its insect hosts mutualism with bacterial endosymbiont Xenorhabdus griffiniae . Through whole-genome re-sequencing genetic mapping we have first time molecularly identified gene responsible mutationally defined phenotypic locus in an nematode. In process observed unexpected mutational spectrum following EMS mutagenesis this species. We find that ortholog essential C. elegans peroxidase skpo-2 controls body size shape S. confirmed identification by inactivating using CRISPR-Cas9. propose will accelerate targeting other nematodes used commercially pest control, X-linked males hemizygous loss function can mate, making easily recognized maintained marker use co-CRISPR.

Язык: Английский

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Exploring DNA Repair Mechanisms in Cancer Biology: Critical Insights and Open Questions

Journal of Molecular Biology, Год журнала: 2023, Номер 436(1), С. 168377 - 168377

Опубликована: Ноя. 29, 2023

Язык: Английский

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