Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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Record number 427458
Title CRISPR immunity relies on the consecutive binding and degradation of negatively supercoiled invader DNA by Cascade and Cas3
Author(s) 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.; Vos, W.M. de; Dame, R.T.; Vries, R. de; Brouns, S.J.J.; Oost, J. van der
Source Molecular Cell 46 (2012)5. - ISSN 1097-2765 - p. 595 - 605.
DOI http://dx.doi.org/10.1016/j.molcel.2012.03.018
Department(s) Microbiological Laboratory
VLAG
Publication type Refereed Article in a scientific journal
Publication year 2012
Keyword(s) rna-polymerase - complex - prokaryotes - mechanism - protein - bacteriophage - resistance - sequence - defense - system
Abstract 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
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