| Title |
Directional R-loop formation by the CRISPR-Cas surveillance complex cascade provides efficient off-target site rejection / |
| Authors |
Rutkauskas, Marius ; Šinkūnas, Tomas ; Songailienė, Inga ; Tikhomirova, Maria S ; Šikšnys, Virginijus ; Seidel, Ralf |
| DOI |
10.1016/j.celrep.2015.01.067 |
| Full Text |
|
| Is Part of |
Cell reports.. New York : Elsevier Inc.. 2015, vol. 10, no. 9, p. 1534-1543.. eISSN 2211-1247 |
| Keywords [eng] |
R-loop formation ; CRISPR-Cas systems ; DNA |
| Abstract [eng] |
CRISPR-Cas systems provide bacteria and archaea with adaptive immunity against foreign nucleic acids. In type I CRISPR-Cas systems, invading DNA is detected by a large ribonucleoprotein surveillance complex called Cascade. The crRNA component of Cascade is used to recognize target sites in foreign DNA (protospacers) by formation of an R-loop driven by base-pairing complementarity. Using single-molecule supercoiling experiments with near base-pair resolution, we probe here the mechanism of R-loop formation and detect short-lived R-loop intermediates on off-target sites bearing single mismatches. We show that R-loops propagate directionally starting from the protospacer-adjacent motif (PAM). Upon reaching a mismatch, R-loop propagation stalls and collapses in a length-dependent manner. This unambiguously demonstrates that directional zipping of the R-loop accomplishes efficient target recognition by rapidly rejecting binding to off-target sites with PAM-proximal mutations. R-loops that reach the protospacer end become locked to license DNA degradation by the auxiliary Cas3 nuclease/helicase without further target verification. |
| Published |
New York : Elsevier Inc |
| Type |
Journal article |
| Language |
English |
| Publication date |
2015 |
