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CRISPR-Cas immunity protects prokaryotes against invading genetic elements.sup.1. It uses the highly conserved Cas1-Cas2 complex to establish inheritable memory (spacers).sup.2-5. How Cas1-Cas2 acquires spacers from foreign DNA fragments (prespacers) and integrates them into the CRISPR locus in the correct orientation is unclear.sup.6,7. Here, using the high spatiotemporal resolution of single-molecule fluorescence, we show that Cas1-Cas2 selects precursors of prespacers from DNA in various forms--including single-stranded DNA and partial duplexes--in a manner that depends on both the length of the DNA strand and the presence of a protospacer adjacent motif (PAM) sequence. We also identify DnaQ exonucleases as enzymes that process the Cas1-Cas2-loaded prespacer precursors into mature prespacers of a suitable size for integration. Cas1-Cas2 protects the PAM sequence from maturation, which results in the production of asymmetrically trimmed prespacers and the subsequent integration of spacers in the correct orientation. Our results demonstrate the kinetic coordination of prespacer precursor selection and PAM trimming, providing insight into the mechanisms that underlie the integration of functional spacers in the CRISPR loci. Cas1-Cas2 selects precursor prespacers from DNA fragments in a length- and PAM-sequence-dependent manner, and these precursors are trimmed by DnaQ exonucleases to enable integration into the CRISPR locus in the correct orientation., Author(s): Sungchul Kim [sup.1] , Luuk Loeff [sup.1] , Sabina Colombo [sup.1] , Slobodan Jergic [sup.2] [sup.3] , Stan J. J. Brouns [sup.1] , Chirlmin Joo [sup.1] Author Affiliations: (1) [...]

In all domains of life, DNA synthesis occurs bidirectionally from replication origins. Despite variable rates of replication fork progression, fork convergence often occurs at specific sites (1). Escherichia coli sets a 'replication fork trap' that allows the first arriving fork to enter but not to leave the terminus region (2-5). The trap is set by oppositely oriented Tus-bound Ter sites that block forks on approach from only one direction (3-7). However, the efficiency of fork blockage by Tus-Ter does not exceed 50% in vivo despite its apparent ability to almost permanently arrest replication forks in vitro (8, 9). Here we use data from single-molecule DNA replication assays and structural studies to show that both polarity and fork-arrest efficiency are determined by a competition between rates of Tus displacement and rearrangement of Tus-Ter interactions that leads to blockage of slower moving replisomes by two distinct mechanisms. To our knowledge this is the first example where intrinsic differences in rates of individual replisomes have different biological outcomes., In the circular E. coli chromosome, two replication forks move from the replication origin to converge opposite in a region that contains ten 23-base-pair Ter (termination) sites and the dif [...]

Limited experimental tools are available to study the consequences of collisions between DNA-bound molecular machines. Here, we repurpose a catalytically inactivated Cas9 (dCas9) construct as a generic, novel, targetable protein-DNA roadblock for studying mechanisms underlying enzymatic activities on DNA substrates in vitro. We illustrate the broad utility of this tool by demonstrating replication fork arrest by the specifically bound dCas9-guideRNA complex to arrest viral, bacterial and eukaryotic replication forks in vitro.