Publication Date:
2015-10-28
Description:
Bacteria and archaea generate adaptive immunity against phages and plasmids by integrating foreign DNA of specific 30-40-base-pair lengths into clustered regularly interspaced short palindromic repeat (CRISPR) loci as spacer segments. The universally conserved Cas1-Cas2 integrase complex catalyses spacer acquisition using a direct nucleophilic integration mechanism similar to retroviral integrases and transposases. How the Cas1-Cas2 complex selects foreign DNA substrates for integration remains unknown. Here we present X-ray crystal structures of the Escherichia coli Cas1-Cas2 complex bound to cognate 33-nucleotide protospacer DNA substrates. The protein complex creates a curved binding surface spanning the length of the DNA and splays the ends of the protospacer to allow each terminal nucleophilic 3'-OH to enter a channel leading into the Cas1 active sites. Phosphodiester backbone interactions between the protospacer and the proteins explain the sequence-nonspecific substrate selection observed in vivo. Our results uncover the structural basis for foreign DNA capture and the mechanism by which Cas1-Cas2 functions as a molecular ruler to dictate the sequence architecture of CRISPR loci.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4662619/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉 〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4662619/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Nunez, James K -- Harrington, Lucas B -- Kranzusch, Philip J -- Engelman, Alan N -- Doudna, Jennifer A -- AI070042/AI/NIAID NIH HHS/ -- R01 AI070042/AI/NIAID NIH HHS/ -- Howard Hughes Medical Institute/ -- England -- Nature. 2015 Nov 26;527(7579):535-8. doi: 10.1038/nature15760. Epub 2015 Oct 21.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California 94720, USA. ; Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, California 94720, USA. ; Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Boston, Massachusetts 02115, USA. ; Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115, USA. ; Department of Chemistry, University of California, Berkeley, Berkeley, California 94720, USA. ; Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA. ; Innovative Genomics Initiative, University of California, Berkeley, Berkeley, California 94720, USA. ; Center for RNA Systems Biology, University of California, Berkeley, Berkeley, California 94720, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/26503043" target="_blank"〉PubMed〈/a〉
Keywords:
*Adaptive Immunity
;
Bacteriophage M13/genetics/immunology
;
Base Sequence
;
CRISPR-Associated Proteins/chemistry/*metabolism
;
Catalytic Domain
;
Clustered Regularly Interspaced Short Palindromic Repeats/*genetics
;
Crystallography, X-Ray
;
DNA, Viral/chemistry/*genetics/*immunology/metabolism
;
Escherichia coli/enzymology/genetics/immunology/virology
;
Integrases/chemistry/metabolism
;
Models, Molecular
;
*Virus Integration/genetics/immunology
Print ISSN:
0028-0836
Electronic ISSN:
1476-4687
Topics:
Biology
,
Chemistry and Pharmacology
,
Medicine
,
Natural Sciences in General
,
Physics
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