CRISPR immunity relies on the consecutive binding and degradation of negatively supercoiled invader DNA by Cascade and Cas3
The prokaryotic CRISPR/Cas immune system is based on genomic loci that contain incorporated sequence tags from viruses and plasmids. Using small guide RNA molecules, these sequences act as a memory to reject returning invaders. Both the Cascade ribonucleoprotein complex and the Cas3 nuclease/helicase are required for CRISPR interference in Escherichia coli, but it is unknown how natural target DNA molecules are recognized and neutralized by their combined action. Here we show that Cascade efficiently locates target sequences in negatively supercoiled DNA, but only if these are flanked by a protospacer-adjacent motif (PAM). PAM recognition by Cascade exclusively involves the crRNA-complementary DNA strand. After Cascade-mediated R loop formation, the Cse1 subunit recruits Cas3, which catalyzes nicking of target DNA through its HD-nuclease domain. The target is then progressively unwound and cleaved by the joint ATP-dependent helicase activity and Mg(2+)-dependent HD-nuclease activity of Cas3, leading to complete target DNA degradation and invader neutralization
Main Authors: | , , , , , , , , , , , , , , |
---|---|
Format: | Article/Letter to editor biblioteca |
Language: | English |
Subjects: | bacteriophage, complex, defense, mechanism, prokaryotes, protein, resistance, rna-polymerase, sequence, system, |
Online Access: | https://research.wur.nl/en/publications/crispr-immunity-relies-on-the-consecutive-binding-and-degradation |
Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
id |
dig-wur-nl-wurpubs-427458 |
---|---|
record_format |
koha |
spelling |
dig-wur-nl-wurpubs-4274582024-06-25 Westra, E.R. Erp, P.B.G. Künne, T. Wong, S.P. Staals, R.H.J. Seegers, C.L.C. Bollen, S. Jore, M.M. Semenova, E. Severinov, K. de Vos, W.M. Dame, R.T. de Vries, R. Brouns, S.J.J. van der Oost, J. Article/Letter to editor Molecular Cell 46 (2012) 5 ISSN: 1097-2765 CRISPR immunity relies on the consecutive binding and degradation of negatively supercoiled invader DNA by Cascade and Cas3 2012 The prokaryotic CRISPR/Cas immune system is based on genomic loci that contain incorporated sequence tags from viruses and plasmids. Using small guide RNA molecules, these sequences act as a memory to reject returning invaders. Both the Cascade ribonucleoprotein complex and the Cas3 nuclease/helicase are required for CRISPR interference in Escherichia coli, but it is unknown how natural target DNA molecules are recognized and neutralized by their combined action. Here we show that Cascade efficiently locates target sequences in negatively supercoiled DNA, but only if these are flanked by a protospacer-adjacent motif (PAM). PAM recognition by Cascade exclusively involves the crRNA-complementary DNA strand. After Cascade-mediated R loop formation, the Cse1 subunit recruits Cas3, which catalyzes nicking of target DNA through its HD-nuclease domain. The target is then progressively unwound and cleaved by the joint ATP-dependent helicase activity and Mg(2+)-dependent HD-nuclease activity of Cas3, leading to complete target DNA degradation and invader neutralization en application/pdf https://research.wur.nl/en/publications/crispr-immunity-relies-on-the-consecutive-binding-and-degradation 10.1016/j.molcel.2012.03.018 https://edepot.wur.nl/222749 bacteriophage complex defense mechanism prokaryotes protein resistance rna-polymerase sequence system (c) publisher Wageningen University & Research |
institution |
WUR NL |
collection |
DSpace |
country |
Países bajos |
countrycode |
NL |
component |
Bibliográfico |
access |
En linea |
databasecode |
dig-wur-nl |
tag |
biblioteca |
region |
Europa del Oeste |
libraryname |
WUR Library Netherlands |
language |
English |
topic |
bacteriophage complex defense mechanism prokaryotes protein resistance rna-polymerase sequence system bacteriophage complex defense mechanism prokaryotes protein resistance rna-polymerase sequence system |
spellingShingle |
bacteriophage complex defense mechanism prokaryotes protein resistance rna-polymerase sequence system bacteriophage complex defense mechanism prokaryotes protein resistance rna-polymerase sequence system Westra, E.R. Erp, P.B.G. Künne, T. Wong, S.P. Staals, R.H.J. Seegers, C.L.C. Bollen, S. Jore, M.M. Semenova, E. Severinov, K. de Vos, W.M. Dame, R.T. de Vries, R. Brouns, S.J.J. van der Oost, J. CRISPR immunity relies on the consecutive binding and degradation of negatively supercoiled invader DNA by Cascade and Cas3 |
description |
The prokaryotic CRISPR/Cas immune system is based on genomic loci that contain incorporated sequence tags from viruses and plasmids. Using small guide RNA molecules, these sequences act as a memory to reject returning invaders. Both the Cascade ribonucleoprotein complex and the Cas3 nuclease/helicase are required for CRISPR interference in Escherichia coli, but it is unknown how natural target DNA molecules are recognized and neutralized by their combined action. Here we show that Cascade efficiently locates target sequences in negatively supercoiled DNA, but only if these are flanked by a protospacer-adjacent motif (PAM). PAM recognition by Cascade exclusively involves the crRNA-complementary DNA strand. After Cascade-mediated R loop formation, the Cse1 subunit recruits Cas3, which catalyzes nicking of target DNA through its HD-nuclease domain. The target is then progressively unwound and cleaved by the joint ATP-dependent helicase activity and Mg(2+)-dependent HD-nuclease activity of Cas3, leading to complete target DNA degradation and invader neutralization |
format |
Article/Letter to editor |
topic_facet |
bacteriophage complex defense mechanism prokaryotes protein resistance rna-polymerase sequence system |
author |
Westra, E.R. Erp, P.B.G. Künne, T. Wong, S.P. Staals, R.H.J. Seegers, C.L.C. Bollen, S. Jore, M.M. Semenova, E. Severinov, K. de Vos, W.M. Dame, R.T. de Vries, R. Brouns, S.J.J. van der Oost, J. |
author_facet |
Westra, E.R. Erp, P.B.G. Künne, T. Wong, S.P. Staals, R.H.J. Seegers, C.L.C. Bollen, S. Jore, M.M. Semenova, E. Severinov, K. de Vos, W.M. Dame, R.T. de Vries, R. Brouns, S.J.J. van der Oost, J. |
author_sort |
Westra, E.R. |
title |
CRISPR immunity relies on the consecutive binding and degradation of negatively supercoiled invader DNA by Cascade and Cas3 |
title_short |
CRISPR immunity relies on the consecutive binding and degradation of negatively supercoiled invader DNA by Cascade and Cas3 |
title_full |
CRISPR immunity relies on the consecutive binding and degradation of negatively supercoiled invader DNA by Cascade and Cas3 |
title_fullStr |
CRISPR immunity relies on the consecutive binding and degradation of negatively supercoiled invader DNA by Cascade and Cas3 |
title_full_unstemmed |
CRISPR immunity relies on the consecutive binding and degradation of negatively supercoiled invader DNA by Cascade and Cas3 |
title_sort |
crispr immunity relies on the consecutive binding and degradation of negatively supercoiled invader dna by cascade and cas3 |
url |
https://research.wur.nl/en/publications/crispr-immunity-relies-on-the-consecutive-binding-and-degradation |
work_keys_str_mv |
AT westraer crisprimmunityreliesontheconsecutivebindinganddegradationofnegativelysupercoiledinvaderdnabycascadeandcas3 AT erppbg crisprimmunityreliesontheconsecutivebindinganddegradationofnegativelysupercoiledinvaderdnabycascadeandcas3 AT kunnet crisprimmunityreliesontheconsecutivebindinganddegradationofnegativelysupercoiledinvaderdnabycascadeandcas3 AT wongsp crisprimmunityreliesontheconsecutivebindinganddegradationofnegativelysupercoiledinvaderdnabycascadeandcas3 AT staalsrhj crisprimmunityreliesontheconsecutivebindinganddegradationofnegativelysupercoiledinvaderdnabycascadeandcas3 AT seegersclc crisprimmunityreliesontheconsecutivebindinganddegradationofnegativelysupercoiledinvaderdnabycascadeandcas3 AT bollens crisprimmunityreliesontheconsecutivebindinganddegradationofnegativelysupercoiledinvaderdnabycascadeandcas3 AT joremm crisprimmunityreliesontheconsecutivebindinganddegradationofnegativelysupercoiledinvaderdnabycascadeandcas3 AT semenovae crisprimmunityreliesontheconsecutivebindinganddegradationofnegativelysupercoiledinvaderdnabycascadeandcas3 AT severinovk crisprimmunityreliesontheconsecutivebindinganddegradationofnegativelysupercoiledinvaderdnabycascadeandcas3 AT devoswm crisprimmunityreliesontheconsecutivebindinganddegradationofnegativelysupercoiledinvaderdnabycascadeandcas3 AT damert crisprimmunityreliesontheconsecutivebindinganddegradationofnegativelysupercoiledinvaderdnabycascadeandcas3 AT devriesr crisprimmunityreliesontheconsecutivebindinganddegradationofnegativelysupercoiledinvaderdnabycascadeandcas3 AT brounssjj crisprimmunityreliesontheconsecutivebindinganddegradationofnegativelysupercoiledinvaderdnabycascadeandcas3 AT vanderoostj crisprimmunityreliesontheconsecutivebindinganddegradationofnegativelysupercoiledinvaderdnabycascadeandcas3 |
_version_ |
1813202495992758272 |