Structures, activity and mechanism of the Type IIS restriction endonuclease PaqCI

Kennedy, Madison A.; Hosford, Christopher J.; Azumaya, Caleigh M.; Luyten, Yvette A.; Chen, Minyong; Morgan, Richard D.; Stoddard, Barry L.

doi:10.1093/nar/gkad228

Title: Structures, activity and mechanism of the Type IIS restriction endonuclease PaqCI

Journal Article · 29 March 2023 · Nucleic Acids Research

DOI: https://doi.org/10.1093/nar/gkad228 · OSTI ID:1963966

Kennedy, Madison A. ^[1]; Hosford, Christopher J. ^[2]; Azumaya, Caleigh M. ^[3]; Luyten, Yvette A. ^[4]; Chen, Minyong ^[4]; Morgan, Richard D. ^[4];

^[1]

Fred Hutchinson Cancer Research Center, Seattle, WA (United States)
New England Biolabs, Ipswich, MA (United States); LifeMine Therapeutics, Cambridge, MA (United States)
Fred Hutchinson Cancer Research Center, Seattle, WA (United States); Genetech, South San Francisco, CA (United States)
New England Biolabs, Ipswich, MA (United States)

Type IIS restriction endonucleases contain separate DNA recognition and catalytic domains and cleave their substrates at well-defined distances outside their target sequences. They are employed in biotechnology for a variety of purposes, including the creation of gene-targeting zinc finger and TAL effector nucleases and DNA synthesis applications such as Golden Gate assembly. The most thoroughly studied Type IIS enzyme, FokI, has been shown to require multimerization and engagement with multiple DNA targets for optimal cleavage activity; however, details of how it or similar enzymes forms a DNA-bound reaction complex have not been described at atomic resolution. Here we describe biochemical analyses of DNA cleavage by the Type IIS PaqCI restriction endonuclease and a series of molecular structures in the presence and absence of multiple bound DNA targets. The enzyme displays a similar tetrameric organization of target recognition domains in the absence or presence of bound substrate, with a significant repositioning of endonuclease domains in a trapped DNA-bound complex that is poised to deliver the first of a series of double-strand breaks. PaqCI and FokI share similar structural mechanisms of DNA cleavage, but considerable differences in their domain organization and quaternary architecture, facilitating comparisons between distinct Type IIS enzymes.

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Research Organization:: Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States). Advanced Light Source (ALS)

Sponsoring Organization:: USDOE Office of Science (SC), Basic Energy Sciences (BES); USDOE

Grant/Contract Number:: AC02-05CH11231

OSTI ID:: 1963966

Alternate ID(s):: OSTI ID: 2422738

Journal Information:: Nucleic Acids Research, Vol. 51, Issue 9; ISSN 0305-1048

Publisher:: Oxford University PressCopyright Statement

Country of Publication:: United States

Language:: English

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