Show simple item record

dc.contributor.authorHilbert, Brendan J.
dc.contributor.authorHayes, Janelle A.
dc.contributor.authorStone, Nicholas P.
dc.contributor.authorXu, Rui-Gang
dc.contributor.authorKelch, Brian A
dc.date2022-08-11T08:08:23.000
dc.date.accessioned2022-08-23T15:53:18Z
dc.date.available2022-08-23T15:53:18Z
dc.date.issued2016-10-19
dc.date.submitted2018-06-20
dc.identifier.citation<p>bioRxiv 080440; doi: https://doi.org/10.1101/080440. <a href="https://doi.org/10.1101/080440" target="_blank">Link to preprint on bioRxiv service.</a></p>
dc.identifier.doi10.1101/080440
dc.identifier.urihttp://hdl.handle.net/20.500.14038/29327
dc.description.abstractMany viruses use a powerful terminase motor to pump their genome inside an empty procapsid shell during virus maturation. The large terminase (TerL) protein contains both enzymatic activities necessary for packaging in such viruses: the ATPase that powers DNA translocation and an endonuclease that cleaves the concatemeric genome both at initiation and completion of genome packaging. However, how TerL binds DNA during translocation and cleavage is still mysterious. Here we investigate DNA binding and cleavage using TerL from the thermophilic phage P74-26. We report the structure of the P74-26 TerL nuclease domain, which allows us to model DNA binding in the nuclease active site. We screened a large panel of TerL variants for defects in binding and DNA cleavage, revealing that the ATPase domain is the primary site for DNA binding, and is required for nucleolysis. The nuclease domain is dispensable for DNA binding but residues lining the active site guide DNA for cleavage. Kinetic analysis of nucleolysis suggests flexible tethering of the nuclease domains during DNA cleavage. We propose that interactions with the procapsid shell during DNA translocation conformationally restrict the nuclease domain, inhibiting cleavage; TerL release from the procapsid upon completion of packaging unlocks the nuclease domains to cleave DNA.
dc.language.isoen_US
dc.relation<p>Now published in <em>Nucleic Acids Research</em> doi: <a href="http://dx.doi.org/10.1093/nar/gkw1356" target="_blank">10.1093/nar/gkw1356</a>.</p>
dc.rightsThe copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license.
dc.rights.urihttp://creativecommons.org/licenses/by-nc-nd/4.0/
dc.subjectbiochemistry
dc.subjectATPase
dc.subjectDNA
dc.subjectnuclease
dc.subjectcleavage
dc.subjectnucleolysis
dc.subjectAmino Acids, Peptides, and Proteins
dc.subjectBiochemistry
dc.subjectEnzymes and Coenzymes
dc.subjectNucleic Acids, Nucleotides, and Nucleosides
dc.titleThe large terminase DNA packaging motor grips DNA with its ATPase domain for cleavage by the flexible nuclease domain [preprint]
dc.typePreprint
dc.source.journaltitlebioRxiv
dc.identifier.legacyfulltexthttps://escholarship.umassmed.edu/cgi/viewcontent.cgi?article=2563&amp;context=faculty_pubs&amp;unstamped=1
dc.identifier.legacycoverpagehttps://escholarship.umassmed.edu/faculty_pubs/1553
dc.identifier.contextkey12346811
refterms.dateFOA2022-08-23T15:53:18Z
html.description.abstract<p>Many viruses use a powerful terminase motor to pump their genome inside an empty procapsid shell during virus maturation. The large terminase (TerL) protein contains both enzymatic activities necessary for packaging in such viruses: the ATPase that powers DNA translocation and an endonuclease that cleaves the concatemeric genome both at initiation and completion of genome packaging. However, how TerL binds DNA during translocation and cleavage is still mysterious. Here we investigate DNA binding and cleavage using TerL from the thermophilic phage P74-26. We report the structure of the P74-26 TerL nuclease domain, which allows us to model DNA binding in the nuclease active site. We screened a large panel of TerL variants for defects in binding and DNA cleavage, revealing that the ATPase domain is the primary site for DNA binding, and is required for nucleolysis. The nuclease domain is dispensable for DNA binding but residues lining the active site guide DNA for cleavage. Kinetic analysis of nucleolysis suggests flexible tethering of the nuclease domains during DNA cleavage. We propose that interactions with the procapsid shell during DNA translocation conformationally restrict the nuclease domain, inhibiting cleavage; TerL release from the procapsid upon completion of packaging unlocks the nuclease domains to cleave DNA.</p>
dc.identifier.submissionpathfaculty_pubs/1553
dc.contributor.departmentDepartment of Biochemistry and Molecular Pharmacology


Files in this item

Thumbnail
Name:
080440.full.pdf
Size:
8.188Mb
Format:
PDF

This item appears in the following Collection(s)

Show simple item record

The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license.
Except where otherwise noted, this item's license is described as The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license.