Biennial Report 2011–2012

Department of Protein-Nucleic Acids Interactions
switches its orientation on DNA to match the polarity of the
second strand and then cuts the phosphodiester bond on the
second DNA strand. Surprisingly, we find that an enzyme flip
required for the second DNA strand cleavage occurs without
an excursion into bulk solution, as the same BcnI molecule acts
processively on both DNA strands. We provide evidence that
after cleavage of the first DNA strand, BcnI remains associated
with the nicked intermediate and relocates to the opposite
strand by a short range diffusive hopping on DNA.
Structure and molecular mechanisms of
CRISPR/Cas systems: projects overview
Streptococcus thermophilus DGCC7710 strain, for which biological
activity of the CRISPR/Cas system has been directly
demonstrated in a phage challenge assay, contains four distinct
systems: CRISPR1, CRISPR2, CRISPR3 and CRISPR4,
which belong to the three distinct Types. Direct spacer incorporation
activity has been demonstrated for the CRISPR1 and
CRISPR3 systems, with the former being more active. The
CRISPR2 system seems to be disrupted and non-functional,
whilst functional activity of CRISPR4 has not yet been demonstrated.
Cas genes, which are specific to the repeat regions
and fall into different families, are located in close proximity to
the spacer-repeat region and encode proteins that often carry
functional nucleic-acid related domains such as nucleases, helicases,
polymerases and nucleotide binding. We aim to characterize
the functional and biochemical activities of Cas proteins
belonging to the CRISPR1, CRISPR3 and CRISPR4 systems
of S. thermophilus.
RNA-guided DNA endonuclease provides
DNA silencing in the Type II system
Type II CRISPR-Cas systems typically consist of only four
Cas genes. The mechanism for DNA interference provided
by the Type II systems remained to be established. We show
that in the CRISPR3 system of Streptococcus thermophilus (a
model and active Type II CRISPR/Cas system), Cas9 associates
with crRNA to form an effector complex which specifically
cleaves matching target dsDNA. This contrasts sharply
with effector complexes for Type I and Type III systems,
which are multisubunit ribonucleoprotein complexes. We
isolated the Cas9-crRNA complex and demonstrated that it
generates in vitro a double strand break at specific sites in
target DNA molecules that are complementary to crRNA sequences
and bear a short proto-spacer adjacent motif (PAM),
in the direct vicinity of the matching sequence. We show that
DNA cleavage is executed by two distinct active sites (RuvC
and HNH) within Cas9, to generate site-specific nicks on opposite
DNA strands. Sequence specificity of the Cas9-crRNA
complex is dictated by the 42 nt crRNA which includes a
20 nt fragment complementary to the proto-spacer sequence
in the target DNA. All together our data demonstrate that
the Cas9-crRNA complex functions as an RNA-guided endonuclease
with sequence-specific target site recognition and
cleavage through two distinct strand nicks.
Figure 5. CRISPR/Cas systems of S. thermophilus DGCC7710.
CRISPR1 and CRISPR3 systems belong to the TypeII, CRISPR2 to the
TypeIII whilst CRISPR4 belongs to the Type I (E.coli subtype).
Figure 6. The Cas9-crRNA complex functions as an RNA-guided DNA
endonuclease. Guided by the crRNA it finds a specific sequence in the
target DNA and Cas9 protein generates two distinct DNA nicks on
opposing dsDNA strands that match the loaded small interfering crRNA
sequence. Specifically, in the presence of Mg 2+ ions, the signature Cas9
protein nicks each DNA strands 3 nt -upstream of the PAM sequence to
generate blunt DNA ends, through RuvC- and HNH-like active sites that
act on separate DNA strands.
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