Cascade-mediated binding and bending of negatively supercoiled DNA

E.R. Westra, B. Nilges, P.B. Erp, J. van der Oost, R.T. Dame, S.J.J. Brouns

Research output: Contribution to journalEditorialAcademicpeer-review

25 Citations (Scopus)

Abstract

Prokaryotes possess various defense mechanisms against invading DNA. Adaptive defense by CRISPR/Cas relies on incorporation of invader DNA sequences in the host genome. In Escherichia coli, processed transcripts of these incorporated sequences (crRNAs) guide Cascade-mediated invader DNA recognition. ( 1) (-) ( 4) Cascade is a multisubunit ribonucleoprotein complex, consisting of one crRNA and five proteins: Cse1, Cse2, Cas7, Cas5 and Cas6e. ( 1) (, ) ( 2) Cascade-mediated DNA recognition requires a conserved sequence adjacent to the target (protospacer adjacent motif, PAM) and a negatively supercoiled DNA topology. ( 3) (, ) ( 4) While Cse1 carries out PAM recognition, ( 5) the Cascade structure suggests that Cse2 may interact with target DNA in the PAM-distal end of the protospacer. ( 6) Using Electrophoretic Mobility Shift Assays, we here describe the function of the Cse1 and Cse2 subunits in the context of protospacer recognition on negatively supercoiled DNA. While Cse1 is required for nonspecific DNA binding, Cse2 appears to be important for specific binding, presumably by mediating stabilizing interactions with the displaced strand, the R-loop, or both. Furthermore, we performed Scanning Force Microscopy using linearized DNA molecules, which facilitates accurate and reliable measurements of Cascade-mediated bending. This analysis reveals that Cascade binding induces flexibility in the DNA target, most likely due to single stranded DNA regions flanking the R-loop
Original languageEnglish
Pages (from-to)1134-1138
JournalRNA Biology
Volume9
Issue number9
DOIs
Publication statusPublished - 2012

Fingerprint

Superhelical DNA
DNA
Clustered Regularly Interspaced Short Palindromic Repeats
Ribonucleoproteins
Conserved Sequence
Atomic Force Microscopy
Single-Stranded DNA
Electrophoretic Mobility Shift Assay
Genome
Escherichia coli

Keywords

  • crispr-cas systems
  • immune-system
  • structural basis
  • rna
  • bacteria
  • archaea
  • defense
  • interference
  • recognition
  • mechanism

Cite this

Westra, E. R., Nilges, B., Erp, P. B., van der Oost, J., Dame, R. T., & Brouns, S. J. J. (2012). Cascade-mediated binding and bending of negatively supercoiled DNA. RNA Biology, 9(9), 1134-1138. https://doi.org/10.4161/rna.21410
Westra, E.R. ; Nilges, B. ; Erp, P.B. ; van der Oost, J. ; Dame, R.T. ; Brouns, S.J.J. / Cascade-mediated binding and bending of negatively supercoiled DNA. In: RNA Biology. 2012 ; Vol. 9, No. 9. pp. 1134-1138.
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abstract = "Prokaryotes possess various defense mechanisms against invading DNA. Adaptive defense by CRISPR/Cas relies on incorporation of invader DNA sequences in the host genome. In Escherichia coli, processed transcripts of these incorporated sequences (crRNAs) guide Cascade-mediated invader DNA recognition. ( 1) (-) ( 4) Cascade is a multisubunit ribonucleoprotein complex, consisting of one crRNA and five proteins: Cse1, Cse2, Cas7, Cas5 and Cas6e. ( 1) (, ) ( 2) Cascade-mediated DNA recognition requires a conserved sequence adjacent to the target (protospacer adjacent motif, PAM) and a negatively supercoiled DNA topology. ( 3) (, ) ( 4) While Cse1 carries out PAM recognition, ( 5) the Cascade structure suggests that Cse2 may interact with target DNA in the PAM-distal end of the protospacer. ( 6) Using Electrophoretic Mobility Shift Assays, we here describe the function of the Cse1 and Cse2 subunits in the context of protospacer recognition on negatively supercoiled DNA. While Cse1 is required for nonspecific DNA binding, Cse2 appears to be important for specific binding, presumably by mediating stabilizing interactions with the displaced strand, the R-loop, or both. Furthermore, we performed Scanning Force Microscopy using linearized DNA molecules, which facilitates accurate and reliable measurements of Cascade-mediated bending. This analysis reveals that Cascade binding induces flexibility in the DNA target, most likely due to single stranded DNA regions flanking the R-loop",
keywords = "crispr-cas systems, immune-system, structural basis, rna, bacteria, archaea, defense, interference, recognition, mechanism",
author = "E.R. Westra and B. Nilges and P.B. Erp and {van der Oost}, J. and R.T. Dame and S.J.J. Brouns",
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Westra, ER, Nilges, B, Erp, PB, van der Oost, J, Dame, RT & Brouns, SJJ 2012, 'Cascade-mediated binding and bending of negatively supercoiled DNA', RNA Biology, vol. 9, no. 9, pp. 1134-1138. https://doi.org/10.4161/rna.21410

Cascade-mediated binding and bending of negatively supercoiled DNA. / Westra, E.R.; Nilges, B.; Erp, P.B.; van der Oost, J.; Dame, R.T.; Brouns, S.J.J.

In: RNA Biology, Vol. 9, No. 9, 2012, p. 1134-1138.

Research output: Contribution to journalEditorialAcademicpeer-review

TY - JOUR

T1 - Cascade-mediated binding and bending of negatively supercoiled DNA

AU - Westra, E.R.

AU - Nilges, B.

AU - Erp, P.B.

AU - van der Oost, J.

AU - Dame, R.T.

AU - Brouns, S.J.J.

PY - 2012

Y1 - 2012

N2 - Prokaryotes possess various defense mechanisms against invading DNA. Adaptive defense by CRISPR/Cas relies on incorporation of invader DNA sequences in the host genome. In Escherichia coli, processed transcripts of these incorporated sequences (crRNAs) guide Cascade-mediated invader DNA recognition. ( 1) (-) ( 4) Cascade is a multisubunit ribonucleoprotein complex, consisting of one crRNA and five proteins: Cse1, Cse2, Cas7, Cas5 and Cas6e. ( 1) (, ) ( 2) Cascade-mediated DNA recognition requires a conserved sequence adjacent to the target (protospacer adjacent motif, PAM) and a negatively supercoiled DNA topology. ( 3) (, ) ( 4) While Cse1 carries out PAM recognition, ( 5) the Cascade structure suggests that Cse2 may interact with target DNA in the PAM-distal end of the protospacer. ( 6) Using Electrophoretic Mobility Shift Assays, we here describe the function of the Cse1 and Cse2 subunits in the context of protospacer recognition on negatively supercoiled DNA. While Cse1 is required for nonspecific DNA binding, Cse2 appears to be important for specific binding, presumably by mediating stabilizing interactions with the displaced strand, the R-loop, or both. Furthermore, we performed Scanning Force Microscopy using linearized DNA molecules, which facilitates accurate and reliable measurements of Cascade-mediated bending. This analysis reveals that Cascade binding induces flexibility in the DNA target, most likely due to single stranded DNA regions flanking the R-loop

AB - Prokaryotes possess various defense mechanisms against invading DNA. Adaptive defense by CRISPR/Cas relies on incorporation of invader DNA sequences in the host genome. In Escherichia coli, processed transcripts of these incorporated sequences (crRNAs) guide Cascade-mediated invader DNA recognition. ( 1) (-) ( 4) Cascade is a multisubunit ribonucleoprotein complex, consisting of one crRNA and five proteins: Cse1, Cse2, Cas7, Cas5 and Cas6e. ( 1) (, ) ( 2) Cascade-mediated DNA recognition requires a conserved sequence adjacent to the target (protospacer adjacent motif, PAM) and a negatively supercoiled DNA topology. ( 3) (, ) ( 4) While Cse1 carries out PAM recognition, ( 5) the Cascade structure suggests that Cse2 may interact with target DNA in the PAM-distal end of the protospacer. ( 6) Using Electrophoretic Mobility Shift Assays, we here describe the function of the Cse1 and Cse2 subunits in the context of protospacer recognition on negatively supercoiled DNA. While Cse1 is required for nonspecific DNA binding, Cse2 appears to be important for specific binding, presumably by mediating stabilizing interactions with the displaced strand, the R-loop, or both. Furthermore, we performed Scanning Force Microscopy using linearized DNA molecules, which facilitates accurate and reliable measurements of Cascade-mediated bending. This analysis reveals that Cascade binding induces flexibility in the DNA target, most likely due to single stranded DNA regions flanking the R-loop

KW - crispr-cas systems

KW - immune-system

KW - structural basis

KW - rna

KW - bacteria

KW - archaea

KW - defense

KW - interference

KW - recognition

KW - mechanism

U2 - 10.4161/rna.21410

DO - 10.4161/rna.21410

M3 - Editorial

VL - 9

SP - 1134

EP - 1138

JO - RNA Biology

JF - RNA Biology

SN - 1547-6286

IS - 9

ER -