Monomeric PcrA helicase processively unwinds plasmid lengths of DNA in the presence of the initiator protein RepD

The helicase PcrA unwinds DNA during asymmetric replication of plasmids, acting with an initiator protein, in our case RepD. Detailed kinetics of PcrA activity were measured using bulk solution and a single-molecule imaging technique to investigate the oligomeric state of the active helicase complex, its processivity and the mechanism of unwinding. By tethering either DNA or PcrA to a microscope coverslip surface, unwinding of both linear and natural circular plasmid DNA by PcrA/RepD was followed in real-time using total internal reflection fluorescence microscopy. Visualization was achieved using a fluorescent single-stranded DNA-binding protein. The single-molecule data show that PcrA, in combination with RepD, can unwind plasmid lengths of DNA in a single run, and that PcrA is active as a monomer. Although the average rate of unwinding was similar in single-molecule and bulk solution assays, the single-molecule experiments revealed a wide distribution of unwinding speeds by different molecules. The average rate of unwinding was several-fold slower than the PcrA translocation rate on single-stranded DNA, suggesting that DNA unwinding may proceed via a partially passive mechanism. However, the fastest dsDNA unwinding rates measured in the single-molecule unwinding assays approached the PcrA translocation speed measured on ssDNA

The effect of unilateral disruption of the centrifugal visual system on normal eye development in chicks raised under constant light conditions

The centrifugal visual system (CVS) comprises a visually driven isthmic feedback projection to the retina. While its function has remained elusive, we have previously shown that, under otherwise normal conditions, unilateral disconnection of centrifugal neurons in the chick affected eye development, inducing a reduced rate of axial elongation that resulted in a unilateral hyperopia in the eye contralateral to the lesion. Here, we further investigate the role of centrifugal neurons in ocular development in chicks reared in an abnormal visual environment, namely constant light. The baseline ocular phenotype of constant light-reared chicks (n = 8) with intact centrifugal neurons was assessed over a 3-week post-hatch time period and, subsequently, compared to chicks raised in normal diurnal lighting (n = 8). Lesions of the isthmo-optic tract or sham surgeries were performed in another seventeen chicks, all raised under constant light. Ocular phenotyping was performed over a 21-day postoperative period to assess changes in refractive state (streak retinoscopy) and ocular component dimensions (A-scan ultrasonography). A pathway-tracing paradigm was employed to quantify lesion success. Chicks raised in constant light conditions with an intact CVS developed shallower anterior chambers combined with elongated vitreous chambers relative to chicks raised in normal diurnal lighting. Seven days following surgery to disrupt centrifugal neurons, a significant positive correlation between refractive error asymmetry between the eyes and lesion success was evident, characterized by hyperopia in the eye contralateral to the lesion. By 21 days post-surgery, these contralateral eyes had become emmetropic, while ipsilateral eyes had developed relative axial hyperopia. Our results provide further support for the hypothesis that the centrifugal visual system can modulate eye development

An anatomical and functional characterisation of the avian centrifugal visual system: a feedback pathway from the brain to the retina

The centrifugal visual system (CVS) is a feedback pathway of predominantly visual information from the brain to both eyes, but principally to the contralateral retina. The CVS is often considered to be something of a peculiarity, regarded as being specific to birds (Aves). Indeed, so-called ‘higher’ vertebrate species are assumed not to even possess such a centrifugal pathway when, in fact, an efferent projection to the retina has been conclusively demonstrated in all vertebrate groups (including humans). Perhaps this point of view reflects the lack of progress made in the elucidation of function in the bird, the dominant model for CVS research in the 120 years since being first described. In the series of experiments presented here, I have begun to investigate the role of the CVS in the modulation of eye growth. In addition, I have addressed a number of unknowns that exist regarding the midbrain connectivity of the CVS. In a series of four parallel lesion experiments, the centrifugal efferent pathway to the retina was unilaterally disrupted in post hatch chicks, raised under different developmental conditions. Under normal visual conditions but in the absence of centrifugal efferents, eyes contralateral to the lesion developed shorter eyes and moderate, relative hyperopia (long-sightedness). In contrast, under constant light conditions, ipsilateral eyes became significantly shorter than fellow (i.e. contralateral) eyes. Compensation for, and recovery from, plus and minus lens-imposed defocus in contralateral eyes was largely unaffected. Centrifugal efferents emanate from two distinct midbrain populations: the isthmo-optic nucleus (ION) and the surrounding scattered cells within the ectopic area (EA). From experiments using pathway tracing techniques, I have demonstrated that, unlike cells of the ION, EA cells do not receive input from primary visual areas. In addition I present evidence for a possible ‘cross-talk’ pathway between centrifugal cells on either side of the midbrain, and discuss its potential involvement in the normally symmetrical eye growth

Refined measurement of SecA-driven protein secretion reveals that translocation is indirectly coupled to ATP turnover

The universally conserved Sec system is the primary method cells utilize to transport proteins across membranes. Until recently, measuring the activity—a prerequisite for understanding how biological systems work—has been limited to discontinuous protein transport assays with poor time resolution or reported by large, nonnatural tags that perturb the process. The development of an assay based on a split superbright luciferase (NanoLuc) changed this. Here, we exploit this technology to unpick the steps that constitute posttranslational protein transport in bacteria. Under the conditions deployed, the transport of a model preprotein substrate (proSpy) occurs at 200 amino acids (aa) per minute, with SecA able to dissociate and rebind during transport. Prior to that, there is no evidence for a distinct, rate-limiting initiation event. Kinetic modeling suggests that SecA-driven transport activity is best described by a series of large (∼30 aa) steps, each coupled to hundreds of ATP hydrolysis events. The features we describe are consistent with a nondeterministic motor mechanism, such as a Brownian ratchet

The AddAB helicase–nuclease catalyses rapid and processive DNA unwinding using a single Superfamily 1A motor domain

The oligomeric state of Superfamily I DNA helicases is the subject of considerable and ongoing debate. While models based on crystal structures imply that a single helicase core domain is sufficient for DNA unwinding activity, biochemical data from several related enzymes suggest that a higher order oligomeric species is required. In this work we characterize the helicase activity of the AddAB helicase–nuclease, which is involved in the repair of double-stranded DNA breaks in Bacillus subtilis. We show that the enzyme is functional as a heterodimer of the AddA and AddB subunits, that it is a rapid and processive DNA helicase, and that it catalyses DNA unwinding using one single-stranded DNA motor of 3′→5′ polarity located in the AddA subunit. The AddB subunit contains a second putative ATP-binding pocket, but this does not contribute to the observed helicase activity and may instead be involved in the recognition of recombination hotspot sequences

Bulk and single-molecule analysis of a bacterial DNA2-like helicase-nuclease reveals a single-stranded DNA looping motor

DNA2 is an essential enzyme involved in DNA replication and repair in eukaryotes. In a search for homologues of this protein, we identified and characterised Geobacillus stearothermophilus Bad, a bacterial DNA helicase-nuclease with similarity to human DNA2. We show that Bad contains an Fe-S cluster and identify four cysteine residues that are likely to co-ordinate the cluster by analogy to DNA2. The purified enzyme specifically recognises ss-dsDNA junctions and possesses ssDNA-dependent ATPase, ssDNA binding, ssDNA endonuclease, 5' to 3' ssDNA translocase and 5' to 3' helicase activity. Single molecule analysis reveals that Bad is a processive DNA motor capable of moving along DNA for distances of >4 kb at a rate of ∼200 bp per second at room temperature. Interestingly, as reported for the homologous human and yeast DNA2 proteins, the DNA unwinding activity of Bad is cryptic and can be unmasked by inactivating the intrinsic nuclease activity. Strikingly, our experiments show that the enzyme loops DNA while translocating, which is an emerging feature of processive DNA unwinding enzymes. The bacterial Bad enzymes will provide an excellent model system for understanding the biochemical properties of DNA2-like helicase-nucleases and DNA looping motor proteins in general.Wellcome Trust [100401/Z/12/Z to M.D.]; EuropeanResearch Council [681299 to F.M.-H.]; Spanish Min-istry of Economy and Competitiveness [BFU2017-83794-PAEI/FEDER, UE to F.M.-H.]; Comunidad de MadridTec4Bio [S2018/NMT-4443 to F.M.-H.]; NanoBioCancer[Y2018/BIO-4747 to F.M.-H.]. Funding for open accesscharge: Wellcome Trust [100401/Z/12/Z].Peer reviewe

Structures of RecBCD in complex with phage-encoded inhibitor proteins reveal distinctive strategies for evasion of a bacterial immunity hub

Following infection of bacterial cells, bacteriophage modulate double-stranded DNA break repair pathways to protect themselves from host immunity systems and prioritise their own recombinases. Here, we present biochemical and structural analysis of two phage proteins, gp5.9 and Abc2, which target the DNA break resection complex RecBCD. These exemplify two contrasting mechanisms for control of DNA break repair in which the RecBCD complex is either inhibited or co-opted for the benefit of the invading phage. Gp5.9 completely inhibits RecBCD by preventing it from binding to DNA. The RecBCD-gp5.9 structure shows that gp5.9 acts by substrate mimicry, binding predominantly to the RecB arm domain and competing sterically for the DNA binding site. Gp5.9 adopts a parallel coiled-coil architecture that is unprecedented for a natural DNA mimic protein. In contrast, binding of Abc2 does not substantially affect the biochemical activities of isolated RecBCD. The RecBCD-Abc2 structure shows that Abc2 binds to the Chi-recognition domains of the RecC subunit in a position that might enable it to mediate the loading of phage recombinases onto its single-stranded DNA products