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  1. Article ; Online: Measurement of Homologous Recombination at Stalled Mammalian Replication Forks.

    Willis, Nicholas A / Scully, Ralph

    Methods in molecular biology (Clifton, N.J.)

    2020  Volume 2153, Page(s) 329–353

    Abstract: Site-specific replication fork barriers (RFBs) have proven valuable tools for studying mechanisms of repair at sites of replication fork stalling in prokaryotes and yeasts. We adapted the Escherichia coli Tus-Ter RFB for use in mammalian cells and used ... ...

    Abstract Site-specific replication fork barriers (RFBs) have proven valuable tools for studying mechanisms of repair at sites of replication fork stalling in prokaryotes and yeasts. We adapted the Escherichia coli Tus-Ter RFB for use in mammalian cells and used it to trigger site-specific replication fork stalling and homologous recombination (HR) at a defined chromosomal locus in mammalian cells. By comparing HR responses induced at the Tus-Ter RFB with those induced by a site-specific double-strand break (DSB), we have begun to uncover how the mechanisms of mammalian stalled fork repair differ from those underlying the repair of a replication-independent DSB. Here, we outline how to transiently express the Tus protein in mES cells, how to use flow cytometry to score conservative and aberrant repair outcomes, and how to quantify distinct repair outcomes in response to replication fork stalling at the inducible Tus-Ter chromosomal RFB.
    MeSH term(s) Animals ; Cells, Cultured ; DNA Breaks, Double-Stranded ; DNA Replication ; DNA-Binding Proteins/genetics ; DNA-Binding Proteins/metabolism ; Escherichia coli/genetics ; Escherichia coli/metabolism ; Escherichia coli Proteins/genetics ; Escherichia coli Proteins/metabolism ; Flow Cytometry ; Homologous Recombination ; Mice ; Mouse Embryonic Stem Cells/chemistry ; Mouse Embryonic Stem Cells/cytology ; Transfection
    Chemical Substances DNA replication terminus site-binding protein, E coli ; DNA-Binding Proteins ; Escherichia coli Proteins ; tus protein, E coli
    Language English
    Publishing date 2020-08-24
    Publishing country United States
    Document type Comparative Study ; Journal Article
    ISSN 1940-6029
    ISSN (online) 1940-6029
    DOI 10.1007/978-1-0716-0644-5_23
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  2. Article ; Online: Exploiting CRISPR/Cas9 to engineer precise segmental deletions in mouse embryonic stem cells.

    Elango, Rajula / Panday, Arvind / Willis, Nicholas A / Scully, Ralph

    STAR protocols

    2022  Volume 3, Issue 3, Page(s) 101551

    Abstract: In this protocol, we use CRISPR/Cas9 to generate large deletions of the entire coding region of a gene of interest, generating a hemizygous cell line. Next, we systematically engineer precise in-frame deletions within the intact wild-type allele, ... ...

    Abstract In this protocol, we use CRISPR/Cas9 to generate large deletions of the entire coding region of a gene of interest, generating a hemizygous cell line. Next, we systematically engineer precise in-frame deletions within the intact wild-type allele, facilitating study of multi-domain proteins. The optimized protocol described here allows us to rapidly screen for effective sgRNA pairs and to engineer either an in-frame deletion or a frameshift mutation in high frequencies in mouse embryonic stem cells. For complete details on the use and execution of this protocol, please refer to Panday et al. (2021).
    MeSH term(s) Animals ; CRISPR-Cas Systems/genetics ; Mice ; Mouse Embryonic Stem Cells ; Sequence Deletion
    Language English
    Publishing date 2022-08-19
    Publishing country United States
    Document type Journal Article ; Research Support, Non-U.S. Gov't ; Research Support, N.I.H., Extramural
    ISSN 2666-1667
    ISSN (online) 2666-1667
    DOI 10.1016/j.xpro.2022.101551
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  3. Article ; Online: A modified CUT&RUN-seq technique for qPCR analysis of chromatin-protein interactions.

    Panday, Arvind / Elango, Rajula / Willis, Nicholas A / Scully, Ralph

    STAR protocols

    2022  Volume 3, Issue 3, Page(s) 101529

    Abstract: Chromatin immunoprecipitation coupled with quantitative PCR (ChIP-qPCR) even with optimization may give low signal-to-background ratio and spatial resolution. Here, we adapted Cleavage Under Targets and Release Using Nuclease (CUT&RUN) (originally ... ...

    Abstract Chromatin immunoprecipitation coupled with quantitative PCR (ChIP-qPCR) even with optimization may give low signal-to-background ratio and spatial resolution. Here, we adapted Cleavage Under Targets and Release Using Nuclease (CUT&RUN) (originally developed by the Henikoff group) to develop CUT&RUN-qPCR. By studying the recruitment of selected proteins (but amenable to other proteins), we find that CUT&RUN-qPCR is more sensitive and gives better spatial resolution than ChIP-qPCR. For complete details on the use and execution of this protocol, please refer to Skene et al. (2018) and Skene and Henikoff (2017).
    MeSH term(s) Chromatin/genetics ; Chromatin Immunoprecipitation/methods ; Chromosomes/metabolism ; Endonucleases ; Micrococcal Nuclease/metabolism
    Chemical Substances Chromatin ; Endonucleases (EC 3.1.-) ; Micrococcal Nuclease (EC 3.1.31.1)
    Language English
    Publishing date 2022-07-31
    Publishing country United States
    Document type Journal Article ; Research Support, Non-U.S. Gov't ; Research Support, N.I.H., Extramural
    ISSN 2666-1667
    ISSN (online) 2666-1667
    DOI 10.1016/j.xpro.2022.101529
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  4. Article ; Online: A modified CUT&RUN-seq technique for qPCR analysis of chromatin-protein interactions

    Arvind Panday / Rajula Elango / Nicholas A. Willis / Ralph Scully

    STAR Protocols, Vol 3, Iss 3, Pp 101529- (2022)

    2022  

    Abstract: Summary: Chromatin immunoprecipitation coupled with quantitative PCR (ChIP-qPCR) even with optimization may give low signal-to-background ratio and spatial resolution. Here, we adapted Cleavage Under Targets and Release Using Nuclease (CUT&RUN) ( ... ...

    Abstract Summary: Chromatin immunoprecipitation coupled with quantitative PCR (ChIP-qPCR) even with optimization may give low signal-to-background ratio and spatial resolution. Here, we adapted Cleavage Under Targets and Release Using Nuclease (CUT&RUN) (originally developed by the Henikoff group) to develop CUT&RUN-qPCR. By studying the recruitment of selected proteins (but amenable to other proteins), we find that CUT&RUN-qPCR is more sensitive and gives better spatial resolution than ChIP-qPCR.For complete details on the use and execution of this protocol, please refer to Skene et al. (2018) and Skene and Henikoff (2017). : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.
    Keywords Cell Biology ; Cell-based Assays ; Molecular Biology ; Chromatin immunoprecipitation (ChIP) ; Science (General) ; Q1-390
    Language English
    Publishing date 2022-09-01T00:00:00Z
    Publisher Elsevier
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  5. Article ; Online: Recombination and restart at blocked replication forks.

    Scully, Ralph / Elango, Rajula / Panday, Arvind / Willis, Nicholas A

    Current opinion in genetics & development

    2021  Volume 71, Page(s) 154–162

    Abstract: Replication fork stalling occurs when the replisome encounters a barrier to normal fork progression. Replisome stalling events are common during scheduled DNA synthesis, but vary in their severity. At one extreme, a lesion may induce only temporary ... ...

    Abstract Replication fork stalling occurs when the replisome encounters a barrier to normal fork progression. Replisome stalling events are common during scheduled DNA synthesis, but vary in their severity. At one extreme, a lesion may induce only temporary pausing of a DNA polymerase; at the other, it may present a near-absolute barrier to the replicative helicase and effectively block fork progression. Many alternative pathways have evolved to respond to these different types of replication stress. Among these, the homologous recombination (HR) pathway plays an important role, protecting the stalled fork and processing it for repair. Here, we review recent advances in our understanding of how blocked replication forks in vertebrate cells can be processed for recombination and for replication restart.
    MeSH term(s) Chromosomes ; DNA Helicases/genetics ; DNA Replication/genetics
    Chemical Substances DNA Helicases (EC 3.6.4.-)
    Language English
    Publishing date 2021-08-28
    Publishing country England
    Document type Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't ; Review
    ZDB-ID 1077312-5
    ISSN 1879-0380 ; 0959-437X
    ISSN (online) 1879-0380
    ISSN 0959-437X
    DOI 10.1016/j.gde.2021.08.003
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    Zs.A 3240: Show issues Location:
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  6. Article ; Online: Epistatic relationships in the BRCA1-BRCA2 pathway.

    Scully, Ralph

    PLoS genetics

    2011  Volume 7, Issue 7, Page(s) e1002183

    MeSH term(s) Animals ; BRCA2 Protein/genetics ; Epistasis, Genetic ; Homologous Recombination ; Rad51 Recombinase/genetics
    Chemical Substances BRCA2 Protein ; Rad51 Recombinase (EC 2.7.7.-)
    Language English
    Publishing date 2011-07-14
    Publishing country United States
    Document type Journal Article ; Comment
    ZDB-ID 2186725-2
    ISSN 1553-7404 ; 1553-7390
    ISSN (online) 1553-7404
    ISSN 1553-7390
    DOI 10.1371/journal.pgen.1002183
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  7. Article: Two-ended recombination at a Flp-nickase-broken replication fork.

    Elango, Rajula / Nilavar, Namrata / Li, Andrew G / Duffey, Erin E / Jiang, Yuning / Nguyen, Daniel / Abakir, Abdulkadir / Willis, Nicholas A / Houseley, Jonathan / Scully, Ralph

    bioRxiv : the preprint server for biology

    2024  

    Abstract: Collision of a replication fork with a DNA nick is thought to generate a one-ended break, fostering genomic instability. Collision of the opposing converging fork with the nick could, in principle, form a second DNA end, enabling conservative repair by ... ...

    Abstract Collision of a replication fork with a DNA nick is thought to generate a one-ended break, fostering genomic instability. Collision of the opposing converging fork with the nick could, in principle, form a second DNA end, enabling conservative repair by homologous recombination (HR). To study mechanisms of nickase-induced HR, we developed the Flp recombinase "step arrest" nickase in mammalian cells. Flp-nickase-induced HR entails two-ended, BRCA2/RAD51-dependent short tract gene conversion (STGC), BRCA2/RAD51-independent long tract gene conversion, and discoordinated two-ended invasions. HR induced by a replication-independent break and by the Flp-nickase differ in their dependence on
    Language English
    Publishing date 2024-04-10
    Publishing country United States
    Document type Preprint
    DOI 10.1101/2024.04.10.588130
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  8. Article ; Online: The spindle-assembly checkpoint, aneuploidy, and gastrointestinal cancer.

    Scully, Ralph

    The New England journal of medicine

    2010  Volume 363, Issue 27, Page(s) 2665–2666

    MeSH term(s) Aneuploidy ; Gastrointestinal Neoplasms/genetics ; Genomic Instability ; Homozygote ; Humans ; Mutation ; Protein-Serine-Threonine Kinases/genetics ; Spindle Apparatus/genetics
    Chemical Substances Bub1 spindle checkpoint protein (EC 2.7.11.1) ; Protein-Serine-Threonine Kinases (EC 2.7.11.1)
    Language English
    Publishing date 2010-12-29
    Publishing country United States
    Document type Comment ; Editorial
    ZDB-ID 207154-x
    ISSN 1533-4406 ; 0028-4793
    ISSN (online) 1533-4406
    ISSN 0028-4793
    DOI 10.1056/NEJMe1008017
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    Ua VI Zs.34: Show issues Location:
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  9. Article ; Online: A histone code for DNA repair.

    Scully, Ralph

    Nature reviews. Molecular cell biology

    2010  Volume 11, Issue 3, Page(s) 164

    MeSH term(s) Animals ; Chromatin/genetics ; Chromatin/metabolism ; DNA Breaks, Double-Stranded ; DNA Damage ; DNA Repair ; Electrophoresis, Gel, Two-Dimensional ; Histones/metabolism ; Humans ; Intracellular Signaling Peptides and Proteins/metabolism ; Nuclear Proteins/metabolism ; Phosphorylation/radiation effects ; Trans-Activators/metabolism
    Chemical Substances Chromatin ; Histones ; Intracellular Signaling Peptides and Proteins ; MDC1 protein, human ; MDC1 protein, mouse ; Nuclear Proteins ; Trans-Activators
    Language English
    Publishing date 2010-03
    Publishing country England
    Document type Journal Article
    ZDB-ID 2031313-5
    ISSN 1471-0080 ; 1471-0072
    ISSN (online) 1471-0080
    ISSN 1471-0072
    DOI 10.1038/nrm2855
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    Zs.A 5446: Show issues Location:
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    Zs.MG 26: Show issues

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  10. Article ; Online: DNA Polymerase θ: Duct Tape and Zip Ties for a Fragile Genome.

    Willis, Nicholas A / Scully, Ralph

    Molecular cell

    2016  Volume 63, Issue 4, Page(s) 542–544

    Abstract: Using a combination of genetics and cellular DNA rejoining assays, in this issue of Molecular Cell, Wyatt et al. (2016) demonstrate a critical role for mammalian DNA polymerase θ in the rejoining of DNA ends that are poor substrates for classical non- ... ...

    Abstract Using a combination of genetics and cellular DNA rejoining assays, in this issue of Molecular Cell, Wyatt et al. (2016) demonstrate a critical role for mammalian DNA polymerase θ in the rejoining of DNA ends that are poor substrates for classical non-homologous end joining.
    MeSH term(s) Animals ; DNA/genetics ; DNA End-Joining Repair ; DNA Repair ; DNA-Directed DNA Polymerase/genetics ; Humans ; DNA Polymerase theta
    Chemical Substances DNA (9007-49-2) ; DNA-Directed DNA Polymerase (EC 2.7.7.7)
    Language English
    Publishing date 2016-09-16
    Publishing country United States
    Document type Journal Article ; Comment
    ZDB-ID 1415236-8
    ISSN 1097-4164 ; 1097-2765
    ISSN (online) 1097-4164
    ISSN 1097-2765
    DOI 10.1016/j.molcel.2016.08.006
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