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dc.contributor.authorValledor, Melvys
dc.contributor.authorMyers, Richard S.
dc.contributor.authorSchiller, Paul C.
dc.date2022-08-11T08:09:51.000
dc.date.accessioned2022-08-23T16:45:50Z
dc.date.available2022-08-23T16:45:50Z
dc.date.issued2018-08-15
dc.date.submitted2018-10-04
dc.identifier.citation<p>PLoS One. 2018 Aug 15;13(8):e0200955. doi: 10.1371/journal.pone.0200955. eCollection 2018. <a href="https://doi.org/10.1371/journal.pone.0200955">Link to article on publisher's site</a></p>
dc.identifier.issn1932-6203 (Linking)
dc.identifier.doi10.1371/journal.pone.0200955
dc.identifier.pmid30110337
dc.identifier.urihttp://hdl.handle.net/20.500.14038/40759
dc.description.abstractRecombineering has transformed functional genomic analysis. Genome modification by recombineering using the phage lambda Red homologous recombination protein Beta in Escherichia coli has approached 100% efficiency. While highly efficient in E. coli, recombineering using the Red Synaptase/Exonuclease pair (SynExo) in other organisms declines in efficiency roughly correlating with phylogenetic distance from E. coli. SynExo recombinases are common to double-stranded DNA viruses infecting a variety of organisms, including humans. Human Herpes virus 1 (HHV1) encodes a SynExo comprised of ICP8 synaptase and UL12 exonuclease. In a previous study, the Herpes SynExo was reconstituted in vitro and shown to catalyze a model recombination reaction. Here we describe stimulation of gene targeting to edit a novel fluorescent protein gene in the human genome using ICP8 and compared its efficiency to that of a "humanized" version of Beta protein from phage lambda. ICP8 significantly enhanced gene targeting rates in HEK 293T cells while Beta was not only unable to catalyze recombineering but inhibited gene targeting using endogenous recombination functions, despite both synaptases being well-expressed and localized to the nucleus. This proof of concept encourages developing species-specific SynExo recombinases for genome engineering.
dc.language.isoen_US
dc.relation<p><a href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&list_uids=30110337&dopt=Abstract">Link to Article in PubMed</a></p>
dc.rightsCopyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
dc.rights.urihttp://creativecommons.org/publicdomain/zero/1.0/
dc.subjectGene targeting
dc.subjectRecombinant proteins
dc.subjectDNA recombination
dc.subjectHomologous recombination
dc.subject293T cells
dc.subjectDNA repair
dc.subjectDNA replication
dc.subjectDNA annealing
dc.subjectAmino Acids, Peptides, and Proteins
dc.subjectBacteria
dc.subjectGenetic Phenomena
dc.subjectGenetics and Genomics
dc.subjectViruses
dc.titleHerpes ICP8 protein stimulates homologous recombination in human cells
dc.typeArticle
dc.source.journaltitlePloS one
dc.source.volume13
dc.source.issue8
dc.identifier.legacyfulltexthttps://escholarship.umassmed.edu/cgi/viewcontent.cgi?article=4575&amp;context=oapubs&amp;unstamped=1
dc.identifier.legacycoverpagehttps://escholarship.umassmed.edu/oapubs/3563
dc.identifier.contextkey13014899
refterms.dateFOA2022-08-23T16:45:50Z
html.description.abstract<p>Recombineering has transformed functional genomic analysis. Genome modification by recombineering using the phage lambda Red homologous recombination protein Beta in Escherichia coli has approached 100% efficiency. While highly efficient in E. coli, recombineering using the Red Synaptase/Exonuclease pair (SynExo) in other organisms declines in efficiency roughly correlating with phylogenetic distance from E. coli. SynExo recombinases are common to double-stranded DNA viruses infecting a variety of organisms, including humans. Human Herpes virus 1 (HHV1) encodes a SynExo comprised of ICP8 synaptase and UL12 exonuclease. In a previous study, the Herpes SynExo was reconstituted in vitro and shown to catalyze a model recombination reaction. Here we describe stimulation of gene targeting to edit a novel fluorescent protein gene in the human genome using ICP8 and compared its efficiency to that of a "humanized" version of Beta protein from phage lambda. ICP8 significantly enhanced gene targeting rates in HEK 293T cells while Beta was not only unable to catalyze recombineering but inhibited gene targeting using endogenous recombination functions, despite both synaptases being well-expressed and localized to the nucleus. This proof of concept encourages developing species-specific SynExo recombinases for genome engineering.</p>
dc.identifier.submissionpathoapubs/3563
dc.contributor.departmentDepartment of Neurology
dc.source.pagese0200955


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Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
Except where otherwise noted, this item's license is described as Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.