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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 [...]

DNA lesions stall the replisome and proper resolution of these obstructions is critical for genome stability. Replisomes can directly replicate past a lesion by error-prone translesion synthesis. Alternatively, replisomes can reprime DNA synthesis downstream of the lesion, creating a single-stranded DNA gap that is repaired primarily in an error-free, homology-directed manner. Here we demonstrate how structural changes within the Escherichia coli replisome determine the resolution pathway of lesion-stalled replisomes. This pathway selection is controlled by a dynamic interaction between the proofreading subunit of the replicative polymerase and the processivity clamp, which sets a kinetic barrier to restrict access of translesion synthesis (TLS) polymerases to the primer/template junction. Failure of TLS polymerases to overcome this barrier leads to repriming, which competes kinetically with TLS. Our results demonstrate that independent of its exonuclease activity, the proofreading subunit of the replisome acts as a gatekeeper and influences replication fidelity during the resolution of lesion-stalled replisomes. [ABSTRACT FROM AUTHOR]

High-field pulse electron paramagnetic resonance (EPR) is used to introduce [Gd.sup.3+] spin labeling for nanometer-range distance measurements in proteins. The results demonstrated the application of [Gd.sup.3+] labeling as a viable technique for distance measurements at high fields that features an order of magnitude sensitivity improvement, in terms of protein quantity, over X-band pulse EPR measurements using nitroxide spin labels.

A universal interaction motif is proposed to be one mechanism by which those proteins bind the E. coli sliding camp, at a single site on the diner. The hierarchy of peptide binding may be indicative of a competitive hierarchy for the binding of proteins to Beta(sub 2) in various stages or circumstances of DNA replication and repair.

Synchronizing the convergence of the two-oppositely moving DNA replication machineries at specific termination sites is a tightly coordinated process in bacteria. InEscherichia coli, a “replication fork trap” – found within a chromosomal region where forks are allowed to enter but not leave – is set by the protein–DNA roadblock Tus–Ter. The exact sequence of events by which Tus–Terblocks replisomes approaching from one direction but not the other has been the subject of controversy for many decades. Specific protein–protein interactions between the nonpermissive face of Tus and the approaching helicase were challenged by biochemical and structural studies. These studies show that it is the helicase-induced strand separation that triggers the formation of new Tus–Terinteractions at the nonpermissive face – interactions that result in a highly stable “locked” complex. This controversy recently gained renewed attention as three single-molecule-based studies scrutinized this elusive Tus–Termechanism – leading to new findings and refinement of existing models, but also generating new questions. Here, we discuss and compare the findings of each of the single-molecule studies to find their common ground, pinpoint the crucial differences that remain, and push the understanding of this bipartite DNA–protein system further. [ABSTRACT FROM PUBLISHER]