PRIMPOL ready, set, reprime!

Tirman, Stephanie; Cybulla, Emily; Quinet, Annabel; Meroni, Alice; Vindigni, Alessandro

doi:10.1080/10409238.2020.1841089

Cited by 29 publications

(21 citation statements)

References 127 publications

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“…While the previous experiments were done by over-expressing a mutant MFN2 cDNA, the following set of experiments tested genome editing of an endogenous gene. First, we used an existing U2OS line with mutations in the mitochondrial primase PRIMPOL 40 and found that it alters mitochondrial morphology. We followed the Raft-Seq process described above, using the mutated line and a wild type U2OS line as the two labeled populations ( Figure 6a ).…”

Section: Resultsmentioning

confidence: 99%

“…While the previous experiments were done by over-expressing a mutant MFN2 cDNA, we also sought to study editing of an endogenous gene. As a proof of concept for endogenous mutations, we first used an existing U2OS line with mutations in the mitochondrial primase PRIMPOL 42 and found that it alters mitochondrial morphology. We then followed the Raft-Seq process described above and verified that the platform performs well for an endogenous genetic perturbation (AUC 0.90, data not shown).…”

Section: Resultsmentioning

confidence: 99%

Section: Scanning Mutagenesis With Mfn2 Vusmentioning

confidence: 99%

See 2 more Smart Citations

Mitochondrial Phenotypes Distinguish Pathogenic MFN2 Mutations by Pooled Functional Genomics Screen

Yenkin

Kremitzki

et al. 2021

Preprint

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Most human genetic variation is classified as VUS - variants of uncertain significance. While advances in genome editing have allowed innovation in pooled screening platforms, many screens deal with relatively simple readouts (viability, fluorescence) and cannot identify the complex cellular phenotypes that underlie most human diseases. In this paper, we present a generalizable functional genomics platform that combines high-content imaging, machine learning, and microraft isolation in a new method termed "Raft-Seq". We highlight the efficacy of our platform by showing its ability to distinguish pathogenic point mutations of the mitochondrial regulator MFN2, even when the cellular phenotype is subtle. We also show that our platform achieves its efficacy using multiple cellular features, which can be configured on-the-fly. Raft-Seq enables a new way to perform pooled screening on sets of mutations in biologically relevant cells, with the ability to physically capture any cell with a perturbed phenotype and expand it clonally, directly from the primary screen.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Scanning Mutagenesis With Mfn2 Vusmentioning

confidence: 99%

See 1 more Smart Citation

Mitochondrial Phenotypes Distinguish Pathogenic MFN2 Mutations by Pooled Functional Genomics Screen

Yenkin

Kremitzki

et al. 2021

Preprint

View full text Add to dashboard Cite

show abstract

“…While the previous experiments were done by over-expressing a mutant MFN2 cDNA, we also sought to study editing of an endogenous gene. As a proof of concept for endogenous mutations, we rst used an existing U2OS line with mutations in the mitochondrial primase PRIMPOL 42 and found that it alters mitochondrial morphology. We then followed the Raft-Seq process described above and veri ed that the platform performs well for an endogenous genetic perturbation (AUC 0.90, data not shown).…”

Section: Scanning Mutagenesis With Mfn2 Vusmentioning

confidence: 99%

Mitochondrial Phenotypes Distinguish Pathogenic MFN2 Mutations by Pooled Functional Genomics Screen

Yenkin

Bramley

Waligorski

et al. 2022

Preprint

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Most human genetic variation is classified as VUS - variants of uncertain significance. While advances in genome editing have allowed innovation in pooled screening platforms, many screens deal with relatively simple readouts (viability, fluorescence) and cannot identify the complex cellular phenotypes that underlie most human diseases. In this paper, we present a generalizable functional genomics platform that combines high-content imaging, machine learning, and microraft isolation in a new method termed “Raft-Seq”. We highlight the efficacy of our platform by showing its ability to distinguish pathogenic point mutations of the mitochondrial regulator MFN2, even when the cellular phenotype is subtle. We also show that our platform achieves its efficacy using multiple cellular features, which can be configured on-the-fly. Raft-Seq enables a new way to perform pooled screening on sets of mutations in biologically relevant cells, with the ability to physically capture any cell with a perturbed phenotype and expand it clonally, directly from the primary screen.

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“…Recent observations have suggested that DNA lesion bypasses on the leading strand can be fulfilled by a newly discovered TLS Pol called Primpol. This enzyme has the unique ability to bypass DNA lesions as well as to reprime DNA synthesis downstream of the lesion [17]. Its recruitment depends upon the ssDNA binding protein RPA [18], and can therefore readily occur on the long stretches of ssDNA generated on the leading strand upon replication fork uncoupling.…”

Section: Recruitment and Action Of Tls Dna Polymerasesmentioning

confidence: 99%

Translesion Synthesis or Repair by Specialized DNA Polymerases Limits Excessive Genomic Instability upon Replication Stress

Maiorano

Etri

Franchet

et al. 2021

IJMS

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DNA can experience “replication stress”, an important source of genome instability, induced by various external or endogenous impediments that slow down or stall DNA synthesis. While genome instability is largely documented to favor both tumor formation and heterogeneity, as well as drug resistance, conversely, excessive instability appears to suppress tumorigenesis and is associated with improved prognosis. These findings support the view that karyotypic diversity, necessary to adapt to selective pressures, may be limited in tumors so as to reduce the risk of excessive instability. This review aims to highlight the contribution of specialized DNA polymerases in limiting extreme genetic instability by allowing DNA replication to occur even in the presence of DNA damage, to either avoid broken forks or favor their repair after collapse. These mechanisms and their key regulators Rad18 and Polθ not only offer diversity and evolutionary advantage by increasing mutagenic events, but also provide cancer cells with a way to escape anti-cancer therapies that target replication forks.

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PRIMPOL ready, set, reprime!

Cited by 29 publications

References 127 publications

Mitochondrial Phenotypes Distinguish Pathogenic MFN2 Mutations by Pooled Functional Genomics Screen

Mitochondrial Phenotypes Distinguish Pathogenic MFN2 Mutations by Pooled Functional Genomics Screen

Mitochondrial Phenotypes Distinguish Pathogenic MFN2 Mutations by Pooled Functional Genomics Screen

Translesion Synthesis or Repair by Specialized DNA Polymerases Limits Excessive Genomic Instability upon Replication Stress

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