Binding partners regulate unfolding of myosin VI to activate the molecular motor

Santos, Ália dos; Fili,; Hari-Gupta,; Gough,; Wang, Hong; Martin-Fernandez,; Arron,; Waite, David W.; Chew, Fook Tim; Toseland, Christopher P.

doi:10.1101/2020.05.10.079236

Cited by 4 publications

(4 citation statements)

References 47 publications

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“…To further address this point, we performed single molecule localisation microscopy (STORM) analysis with the 50A guide 2 h measurement ( Figure 4 G). This approach is equivalent to the widefield imaging performed in Figure 2 and Figure 3 , but with approximately five-fold increased resolution and the ability to count single molecules and quantify clusters [ 21 , 22 ]. We detected γH2AX foci and then quantified the foci using cluster analysis ( Figure 4 H).…”

Section: Resultsmentioning

confidence: 99%

A Targeted and Tuneable DNA Damage Tool Using CRISPR/Cas9

et al. 2021

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Mammalian cells are constantly subjected to a variety of DNA damaging events that lead to the activation of DNA repair pathways. Understanding the molecular mechanisms of the DNA damage response allows the development of therapeutics which target elements of these pathways. Double-strand breaks (DSB) are particularly deleterious to cell viability and genome stability. Typically, DSB repair is studied using DNA damaging agents such as ionising irradiation or genotoxic drugs. These induce random lesions at non-predictive genome sites, where damage dosage is difficult to control. Such interventions are unsuitable for studying how different DNA damage recognition and repair pathways are invoked at specific DSB sites in relation to the local chromatin state. The RNA-guided Cas9 (CRISPR-associated protein 9) endonuclease enzyme is a powerful tool to mediate targeted genome alterations. Cas9-based genomic intervention is attained through DSB formation in the genomic area of interest. Here, we have harnessed the power to induce DSBs at defined quantities and locations across the human genome, using custom-designed promiscuous guide RNAs, based on in silico predictions. This was achieved using electroporation of recombinant Cas9-guide complex, which provides a generic, low-cost and rapid methodology for inducing controlled DNA damage in cell culture models.

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Section: Resultsmentioning

confidence: 99%

A Targeted and Tuneable DNA Damage Tool Using CRISPR/Cas9

et al. 2021

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“…Previous work showed that full-length NDP52 is mainly a dimer in solution 6,9 . To confirm this, we used Size-Exclusion Chromatography with Multi-Angle Light Scattering (SEC-MALS).…”

Section: Ndp52 Oligomerisation and Structurementioning

confidence: 98%

“…At the C-terminal, ZF1 has been characterised as an unconventional dynamic zinc finger, whilst ZF2 is a canonical C2H2-type zinc finger 7 . The C-terminal domains of NDP52 are responsible for interactions with ubiquitin, which allows binding to ubiquitylated pathogens, as well as interactions with actin-based motor Myosin VI (MVI) 3,[7][8][9] . In the cytoplasm, interactions between NDP52 and MVI allow autophagosome maturation 5 .…”

Section: Introductionmentioning

confidence: 99%

Autophagy receptor NDP52 alters DNA conformation to modulate RNA Polymerase II transcription

Santos

Rollins

Hari-Gupta

et al. 2022

Preprint

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NDP52 is an autophagy receptor involved in the recognition and degradation of invading pathogens and damaged organelles. Although NDP52 was first identified in the nucleus and is expressed throughout the cell, to date, there is no clear nuclear function for NDP52. Here, we use a multidisciplinary approach to characterise the biochemical properties and nuclear roles of NDP52. We found that NDP52 clusters with RNA Polymerase II (RNAPII) at transcription initiation sites and that its overexpression promotes the formation of additional transcriptional clusters. We also show that depletion of NDP52 impacts overall gene-expression levels in two model mammalian cells, and that transcription inhibition affects the spatial organisation and molecular dynamics of NDP52 in the nucleus. This directly links NDP52 to a role in RNAPII-dependent transcription. Furthermore, we also show that NDP52 binds specifically and with high affinity to double-stranded DNA (dsDNA) and that this interaction leads to changes in DNA structure in vitro. This, together with our proteomics data indicating enrichment for interactions with nucleosome remodelling proteins and DNA structure regulators, suggests a possible function for NDP52 in chromatin regulation. Overall, here we uncover novel nuclear roles for NDP52 in gene expression and DNA structure regulation.

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“…To further address this point, we performed single molecule localisation microscopy (STORM) analysis with the 50A guide 2-hour measurement ( Figure 4G). This approach is equivalent to the widefield imaging performed in Figure 2 and 3, but with approximately five-fold increased resolution and the ability to count single molecules and quantify clusters 18,19 . We detected gH2AX foci and then quantified the foci using cluster analysis ( Figure 4H).…”

Section: Tuning the Number Of Dna Damage Sites Using Cas9 And Promiscmentioning

confidence: 99%

A targeted and tuneable DNA damage tool using CRISPR/Cas9

Emmanouilidis

Fili

Cook

et al. 2021

Preprint

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Mammalian cells are constantly subjected to a variety of DNA damaging events that lead to the activation of DNA repair pathways. Understanding the molecular mechanisms of the DNA damage response allows the development of therapeutics which target elements of these pathways.Double-Strand Breaks (DSB) are particularly deleterious to cell viability and genome stability. Typically, DSB repair is studied using DNA damaging agents such as ionising irradiation or genotoxic drugs. These induce random lesions at non-predictive genome sites, where damage dosage is difficult to control. Such interventions are unsuitable for studying how different DNA damage recognition and repair pathways are invoked at specific DSB sites in relation to the local chromatin state.The RNA-guided Cas9 (CRISPR associated protein 9) endonuclease enzyme, is a powerful tool to mediate targeted genome alterations. Cas9-based genomic intervention is attained through DSB formation in the genomic area of interest. Here, we have harnessed the power to induce DSBs at defined quantities and locations across the human genome, using custom-designed promiscuous guide RNAs, based on in silico predictions. This was achieved using electroporation of recombinant Cas9-guide complex which provides a generic, low-cost and rapid methodology for inducing controlled DNA damage in cell culture models.

show abstract

Binding partners regulate unfolding of myosin VI to activate the molecular motor

Cited by 4 publications

References 47 publications

A Targeted and Tuneable DNA Damage Tool Using CRISPR/Cas9

A Targeted and Tuneable DNA Damage Tool Using CRISPR/Cas9

Autophagy receptor NDP52 alters DNA conformation to modulate RNA Polymerase II transcription

A targeted and tuneable DNA damage tool using CRISPR/Cas9

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