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Abstract HROB promotes the MCM8-9 helicase in DNA damage response. To understand how HROB activates MCM8-9, we defined their interaction interface. We showed that HROB makes important yet transient contacts with both MCM8 and MCM9, and binds the MCM8-9 heterodimer with the highest affinity. MCM8-9-HROB prefer branched DNA structures, and display low DNA unwinding processivity. MCM8-9 unwinds DNA as a hexamer that assembles from dimers on DNA in the presence of ATP. The hexamer involves two repeating protein-protein interfaces between the alternating MCM8 and MCM9 subunits. One of these interfaces is quite stable and forms an obligate heterodimer across which HROB binds. The other interface is labile and mediates hexamer assembly, independently of HROB. The ATPase site formed at the labile interface contributes disproportionally more to DNA unwinding than that at the stable interface. Here, we show that HROB promotes DNA unwinding downstream of MCM8-9 loading and ring formation on ssDNA.

Abstract CRISPR-Cas effector complexes enable the defense against foreign nucleic acids and have recently been exploited as molecular tools for precise genome editing at a target locus. To bind and cleave their target, the CRISPR-Cas effectors have to interrogate the entire genome for the presence of a matching sequence. Here we dissect the target search and recognition process of the Type I CRISPR-Cas complex Cascade by simultaneously monitoring DNA binding and R-loop formation by the complex. We directly quantify the effect of DNA supercoiling on the target recognition probability and demonstrate that Cascade uses facilitated diffusion for its target search. We show that target search and target recognition are tightly linked and that DNA supercoiling and limited 1D diffusion need to be considered when understanding target recognition and target search by CRISPR-Cas enzymes and engineering more efficient and precise variants.

Based on thorough single-molecule analysis of off-target binding by the CRISPR-Cas surveillance complex Cascade, a mechanism-based model is presented that quantitatively describes the recognition dynamics of mutated targets by this protein.

The Mre11-Rad50 (MR) complex has key functions in the detection, signaling and repair of DNA breaks. Here the authors use transmission electron microscopy to show MR oligomerization is governed by a small beta-sheet protruding from the head domain of Rad50 at the base of the MR structure, and reveal MR head domain oligomerization is required for efficient DNA end resection.

An enzymatic ensemble including Dna2 functions in DNA end resection; the function of the single-stranded DNA binding protein RPA in this complex has been underappreciated. Here the authors employ molecular modeling, biochemistry, and single molecule biophysics to reveal RPA directly promotes Dna2 recruitment, nuclease and helicase activities.

Miniature Cas12f editing systems are well suited for in vivo editing applications. Here the authors characterize the intrinsic activity of SpCas12f1 in plant and animal cells.

Summary: The multi-subunit type I CRISPR-Cas surveillance complex Cascade uses its crRNA to recognize dsDNA targets. Recognition involves DNA unwinding and base-pairing between the crRNA spacer region and a complementary DNA strand, resulting in formation of an R-loop structure. The modular Cascade architecture allows assembly of complexes containing crRNAs with altered spacer lengths that promise increased target specificity in emerging biotechnological applications. Here we produce type I-E Cascade complexes containing crRNAs with up to 57-nt-long spacers. We show that these complexes form R-loops corresponding to the designed target length, even for the longest spacers tested. Furthermore, the complexes can bind their targets with much higher affinity compared with the wild-type form. However, target recognition and the subsequent Cas3-mediated DNA cleavage do not require extended R-loops but already occur for wild-type-sized R-loops. These findings set important limits for specificity improvements of type I CRISPR-Cas systems. : Songailiene et al. show that engineered versions of Cascade with elongated crRNAs can base-pair with target DNA over 57 bp. However, target recognition only requires wild-type-sized base-pairing of ∼30-bp length. These findings define constraints for improving the specificity of these complexes in biotechnological applications. Keywords: Cascade, CRISPR, type I-E, single molecule, crRNA, R-loop, Streptococcus thermophilus, Cse2, Cse1, genome engineering

The restricted access of regulatory factors to their binding sites on DNA wrapped around the nucleosomes is generally interpreted in terms of molecular shielding exerted by nucleosomal structure and internucleosomal interactions. Binding of proteins to DNA often includes intercalation of hydrophobic amino acids into the DNA. To assess the role of constrained superhelicity in limiting these interactions, we studied the binding of small molecule intercalators to chromatin in close to native conditions by laser scanning cytometry. We demonstrate that the nucleosome-constrained superhelical configuration of DNA is the main barrier to intercalation. As a result, intercalating compounds are virtually excluded from the nucleosome-occupied regions of the chromatin. Binding of intercalators to extranucleosomal regions is limited to a smaller degree, in line with the existence of net supercoiling in the regions comprising linker and nucleosome free DNA. Its relaxation by inducing as few as a single nick per ~50 kb increases intercalation in the entire chromatin loop, demonstrating the possibility for long-distance effects of regulatory potential.

The cellular prion protein (PrPC) is implicated in neuroprotective signaling and neurotoxic pathways in both prion diseases and Alzheimer's disease (AD). Specifically, the intrinsically disordered N-terminal domain (N-PrP) has been shown to interact with neurotoxic ligands, such as Aβ and Scrapie prion protein (PrPSc), and to be crucial for the neuroprotective activity of PrPC. To gain further insight into cellular pathways tied to PrP, we analyzed the brain interactome of N-PrP. As a novel approach employing recombinantly expressed PrP and intein-mediated protein ligation, we used N-PrP covalently coupled to beads as a bait for affinity purification. N-PrP beads were incubated with human AD or control brain lysates. N-PrP binding partners were then identified by electrospray ionization tandem mass spectrometry (nano ESI-MS/MS). In addition to newly identified proteins we found many previously described PrP interactors, indicating a crucial role of the intrinsically disordered part of PrP in mediating protein interactions. Moreover, some interactors were found only in either non-AD or AD brain, suggesting aberrant PrPC interactions in the pathogenesis of AD.

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.

Human DNA2 (hDNA2) contains both a helicase and a nuclease domain within the same polypeptide. The nuclease of hDNA2 is involved in a variety of DNA metabolic processes. Little is known about the role of the hDNA2 helicase. Using bulk and single-molecule approaches, we show that hDNA2 is a processive helicase capable of unwinding kilobases of dsDNA in length. The nuclease activity prevents the engagement of the helicase by competing for the same substrate, hence prominent DNA unwinding by hDNA2 alone can only be observed using the nuclease-deficient variant. We show that the helicase of hDNA2 functionally integrates with BLM or WRN helicases to promote dsDNA degradation by forming a heterodimeric molecular machine. This collectively suggests that the hDNA2 motor promotes the enzyme's capacity to degrade dsDNA in conjunction with BLM or WRN and thus promote the repair of broken DNA.

In many hyperthermophilic archaea the DNA binding protein TrmBL2 or one of its homologues is abundantly expressed. TrmBL2 is thought to play a significant role in modulating the chromatin architecture in combination with the archaeal histone proteins and Alba. However, its precise physiological role is poorly understood. It has been previously shown that upon binding TrmBL2 covers double-stranded DNA, which leads to the formation of a thick and fibrous filament. Here we investigated the filament formation process as well as the stabilization of DNA by TrmBL2 from Pyroccocus furiosus in detail. We used magnetic tweezers that allow to monitor changes of the DNA mechanical properties upon TrmBL2 binding on the single-molecule level. Extended filaments formed in a cooperative manner and were considerably stiffer than bare double-stranded DNA. Unlike Alba, TrmBL2 did not form DNA cross-bridges. The protein was found to bind double- and single-stranded DNA with similar affinities. In mechanical disruption experiments of DNA hairpins this led to stabilization of both, the double- (before disruption) and the single-stranded (after disruption) DNA forms. Combined, these findings suggest that the biological function of TrmBL2 is not limited to modulating genome architecture and acting as a global repressor but that the protein acts additionally as a stabilizer of DNA secondary structure.