Fast and efficient DNA replication with purified human proteins.

Chromosome replication is performed by a complex and intricate ensemble of proteins termed the replisome, where the DNA polymerases Polδ and Polε, DNA polymerase α-primase (Polα) and accessory proteins including AND-1, CLASPIN and TIMELESS-TIPIN (respectively known as Ctf4, Mrc1 and Tof1-Csm3 in Saccharomyces cerevisiae) are organized around the CDC45-MCM-GINS (CMG) replicative helicase1-7. Because a functional human replisome has not been reconstituted from purified proteins, how these factors contribute to human DNA replication and whether additional proteins are required for optimal DNA synthesis are poorly understood. Here we report the biochemical reconstitution of human replisomes that perform fast and efficient DNA replication using 11 purified human replication factors made from 43 polypeptides. Polε, but not Polδ, is crucial for optimal leading-strand synthesis. Unexpectedly, Polε-mediated leading-strand replication is highly dependent on the sliding-clamp processivity factor PCNA and the alternative clamp loader complex CTF18-RFC. We show how CLASPIN and TIMELESS-TIPIN contribute to replisome progression and demonstrate that, in contrast to the budding yeast replisome8, AND-1 directly augments leading-strand replication. Moreover, although AND-1 binds to Polα9,10, the interaction is dispensable for lagging-strand replication, indicating that Polα is functionally recruited via an AND-1-independent mechanism for priming in the human replisome. Collectively, our work reveals how the human replisome achieves fast and efficient leading-strand and lagging-strand DNA replication, and provides a powerful system for future studies of the human replisome and its interactions with other DNA metabolic processes.

Commercial and business aspects of alpha radioligand therapeutics.

Radioligand therapy (RLT) is gaining traction as a safe and effective targeted approach for the treatment of many cancer types, reflected by a substantial and growing commercial market (valued at $7.78 billion in 2021, with a projected value of $13.07 billion by 2030). Beta-emitting RLTs have a long history of clinical success dating back to the approval of Zevalin and Bexxar in the early 2000s, later followed by Lutathera and Pluvicto. Alpha radioligand therapeutics (ARTs) offer the potential for even greater success. Driven by ground-breaking clinical results in early trials, improved isotope availability, and better understanding of isotope and disease characteristics, the global market for alpha emitters was estimated at $672.3 million for the year 2020, with projected growth to $5.2 billion by 2027. New company formations, promising clinical trial data, and progression for many radioligand therapy products, as well as an inflow of investor capital, are contributing to this expanding field. Future growth will be fueled by further efficacy and safety data from ART clinical trials and real-world results, but challenges remain. Radionuclide supply, manufacturing, and distribution are key obstacles for growth of the field. New models of delivery are needed, along with cross-disciplinary training of specialized practitioners, to ensure patient access and avoid challenges faced by early RLT candidates such as Zevalin and Bexxar. Understanding of the history of radiation medicine is critical to inform what may be important to the success of ART-most past projections were inaccurate and it is important to analyze the reasons for this. Practical considerations in how radiation medicine is delivered and administered are important to understand in order to inform future approaches.

Structure of a human replisome shows the organisation and interactions of a DNA replication machine.

The human replisome is an elaborate arrangement of molecular machines responsible for accurate chromosome replication. At its heart is the CDC45-MCM-GINS (CMG) helicase, which, in addition to unwinding the parental DNA duplex, arranges many proteins including the leading-strand polymerase Pol ε, together with TIMELESS-TIPIN, CLASPIN and AND-1 that have key and varied roles in maintaining smooth replisome progression. How these proteins are coordinated in the human replisome is poorly understood. We have determined a 3.2 Šcryo-EM structure of a human replisome comprising CMG, Pol ε, TIMELESS-TIPIN, CLASPIN and AND-1 bound to replication fork DNA. The structure permits a detailed understanding of how AND-1, TIMELESS-TIPIN and Pol ε engage CMG, reveals how CLASPIN binds to multiple replisome components and identifies the position of the Pol ε catalytic domain. Furthermore, the intricate network of contacts contributed by MCM subunits and TIMELESS-TIPIN with replication fork DNA suggests a mechanism for strand separation.

Nucleotide proofreading functions by nematode RAD51 paralogs facilitate optimal RAD51 filament function.

The RAD51 recombinase assembles as helical nucleoprotein filaments on single-stranded DNA (ssDNA) and mediates invasion and strand exchange with homologous duplex DNA (dsDNA) during homologous recombination (HR), as well as protection and restart of stalled replication forks. Strand invasion by RAD51-ssDNA complexes depends on ATP binding. However, RAD51 can bind ssDNA in non-productive ADP-bound or nucleotide-free states, and ATP-RAD51-ssDNA complexes hydrolyse ATP over time. Here, we define unappreciated mechanisms by which the RAD51 paralog complex RFS-1/RIP-1 limits the accumulation of RAD-51-ssDNA complexes with unfavorable nucleotide content. We find RAD51 paralogs promote the turnover of ADP-bound RAD-51 from ssDNA, in striking contrast to their ability to stabilize productive ATP-bound RAD-51 nucleoprotein filaments. In addition, RFS-1/RIP-1 inhibits binding of nucleotide-free RAD-51 to ssDNA. We propose that 'nucleotide proofreading' activities of RAD51 paralogs co-operate to ensure the enrichment of active, ATP-bound RAD-51 filaments on ssDNA to promote HR.

Dynamics of Replication Fork Progression Following Helicase-Polymerase Uncoupling in Eukaryotes.

Leading-strand polymerase stalling at DNA damage impairs replication fork progression. Using biochemical approaches, we show this arises due to both slower template unwinding following helicase-polymerase uncoupling and establishment of prolonged stalled fork structures. Fork slowing and stalling occur at structurally distinct lesions, are always associated with continued lagging-strand synthesis, are observed when either Pol ε or Pol δ stalls at leading-strand damage, and do not require specific helicase-polymerase coupling factors. Hence, the key trigger for these replisome-intrinsic responses is cessation of leading-strand polymerization, revealing this as a crucial driver of normal replication fork rates. We propose that this helps balance the need for sufficient uncoupling to activate the DNA replication checkpoint with excessive destabilizing single-stranded DNA exposure in eukaryotes.

The Initial Response of a Eukaryotic Replisome to DNA Damage.

The replisome must overcome DNA damage to ensure complete chromosome replication. Here, we describe the earliest events in this process by reconstituting collisions between a eukaryotic replisome, assembled with purified proteins, and DNA damage. Lagging-strand lesions are bypassed without delay, leaving daughter-strand gaps roughly the size of an Okazaki fragment. In contrast, leading-strand polymerase stalling significantly impacts replication fork progression. We reveal that the core replisome itself can bypass leading-strand damage by re-priming synthesis beyond it. Surprisingly, this restart activity is rare, mainly due to inefficient leading-strand re-priming, rather than single-stranded DNA exposure or primer extension. We find several unanticipated mechanistic distinctions between leading- and lagging-strand priming that we propose control the replisome's initial response to DNA damage. Notably, leading-strand restart was specifically stimulated by RPA depletion, which can occur under conditions of replication stress. Our results have implications for pathway choice at stalled forks and priming at DNA replication origins.

A Polar and Nucleotide-Dependent Mechanism of Action for RAD51 Paralogs in RAD51 Filament Remodeling.

Central to homologous recombination in eukaryotes is the RAD51 recombinase, which forms helical nucleoprotein filaments on single-stranded DNA (ssDNA) and catalyzes strand invasion with homologous duplex DNA. Various regulatory proteins assist this reaction including the RAD51 paralogs. We recently discovered that a RAD51 paralog complex from C. elegans, RFS-1/RIP-1, functions predominantly downstream of filament assembly by binding and remodeling RAD-51-ssDNA filaments to a conformation more proficient for strand exchange. Here, we demonstrate that RFS-1/RIP-1 acts by shutting down RAD-51 dissociation from ssDNA. Using stopped-flow experiments, we show that RFS-1/RIP-1 confers this dramatic stabilization by capping the 5' end of RAD-51-ssDNA filaments. Filament end capping propagates a stabilizing effect with a 5'→3' polarity approximately 40 nucleotides along individual filaments. Finally, we discover that filament capping and stabilization are dependent on nucleotide binding, but not hydrolysis by RFS-1/RIP-1. These data define the mechanism of RAD51 filament remodeling by RAD51 paralogs.

Rad51 Paralogs Remodel Pre-synaptic Rad51 Filaments to Stimulate Homologous Recombination.

Repair of DNA double strand breaks by homologous recombination (HR) is initiated by Rad51 filament nucleation on single-stranded DNA (ssDNA), which catalyzes strand exchange with homologous duplex DNA. BRCA2 and the Rad51 paralogs are tumor suppressors and critical mediators of Rad51. To gain insight into Rad51 paralog function, we investigated a heterodimeric Rad51 paralog complex, RFS-1/RIP-1, and uncovered the molecular basis by which Rad51 paralogs promote HR. Unlike BRCA2, which nucleates RAD-51-ssDNA filaments, RFS-1/RIP-1 binds and remodels pre-synaptic filaments to a stabilized, "open," and flexible conformation, in which the ssDNA is more accessible to nuclease digestion and RAD-51 dissociation rate is reduced. Walker box mutations in RFS-1, which abolish filament remodeling, fail to stimulate RAD-51 strand exchange activity, demonstrating that remodeling is essential for RFS-1/RIP-1 function. We propose that Rad51 paralogs stimulate HR by remodeling the Rad51 filament, priming it for strand exchange with the template duplex.

Playing the end game: DNA double-strand break repair pathway choice.

DNA double-strand breaks (DSBs) are highly toxic lesions that can drive genetic instability. To preserve genome integrity, organisms have evolved several DSB repair mechanisms, of which nonhomologous end-joining (NHEJ) and homologous recombination (HR) represent the two most prominent. It has recently become apparent that multiple layers of regulation exist to ensure these repair pathways are accurate and restricted to the appropriate cellular contexts. Such regulation is crucial, as failure to properly execute DSB repair is known to accelerate tumorigenesis and is associated with several human genetic syndromes. Here, we review recent insights into the mechanisms that influence the choice between competing DSB repair pathways, how this is regulated during the cell cycle, and how imbalances in this equilibrium result in genome instability.

Structure and dynamics of MarA-DNA complexes: an NMR investigation.

An unanswered question regarding gene regulation is how certain proteins are capable of binding to DNA with high affinity at specific but highly degenerate consensus sequences. We have investigated the interactions between the Escherichia coli transcription factor, MarA, and its diverse binding sites using NMR techniques. Complete resonance assignments for the backbone of the MarA protein complexed with DNA oligomers corresponding to its binding sites at the mar, fumC, micF and the fpr promoters were obtained. Secondary structure analysis based on chemical shifts reveals that regions identified as helical in the X-ray structure of the MarA-mar complex are present in the solution structure, although some of the helices are less well defined. The chemical shift differences between the four complexes confirm that helix 3 and helix 6 constitute the major DNA-binding elements. However, in striking contrast with the X-ray data: (i) the protein appears to be present in two or more conformations in each of the complexes; (ii) no slowly exchanging N(zeta)H(2) protons (indicative of hydrogen bonded groups) were observed by NMR for the two arginine residues proposed to form crucial hydrogen bonds in the X-ray structure; and (iii) regions at the N terminus, not observed in the X-ray structure, may be involved in DNA-binding. Taken together, the NMR results indicate that MarA in its complexes with DNA target sites is in a highly dynamic state, allowing for small but significant rearrangements of the side-chains and/or backbone to bind to the different DNA sequences.

The AraC transcriptional activators.

The AraC family of bacterial transcriptional activators regulate diverse genetic systems. Recent X-ray diffraction studies show that the monomeric MarA and Rob activators bind to their asymmetric degenerate DNA sites via two different helix-turn-helix elements. Activation by MarA, SoxS or Rob requires a particular orientation of the asymmetric binding sequence (and hence the activator), depending on its distance from the -10 RNAP signal. Genetic studies are beginning to clarify how the activators interact with RNAP. Growing evidence suggests that for the sugar metabolism activators, multiple binding sites upstream of the promoter anchor the activator in a repressing or nonactivating configuration. By interaction with the sugar and/or CRP, the activator is allosterically altered so it can bind a new set of sites that enable it to activate the promoter. Surprisingly, the virulence activator, Rns, must bind to both upstream and downstream sites in order to activate the rns promoter.

Probing the Escherichia coli transcriptional activator MarA using alanine-scanning mutagenesis: residues important for DNA binding and activation.

The MarA transcriptional activator binds to a 20 bp asymmetric degenerate sequence (marbox) located at different positions and orientations within the promoters of the genes of the Escherichia coli mar regulon. Solution of the MarA-marbox X-ray crystallographic structure suggested the presence of base-specific and non-specific interactions between the marbox and two helix-turn-helix (HTH) motifs on the monomeric MarA. Here, we use alanine-scanning mutagenesis and DNA retardation analysis to: (i) evaluate the contacts between MarA and the marboxes of five differently configured mar regulon promoters; (ii) assess the role of conserved hydrophobic amino acid residues for MarA activity; and (iii) identify residues required for RNA polymerase activation. These analyses revealed that the phosphate-backbone contacts and hydrogen bonds with the bases of the marbox are more significant for DNA binding than are the van der Waals interactions. While both N and C-terminal HTH motifs make essential contributions to binding site affinity, MarA is more sensitive to alterations in the N-terminal HTH. In a similar way, the activity of MarA is more sensitive to alterations in the hydrophobic core of this HTH. Solvent-exposed amino acid residues located at many positions on the MarA surface are important for activity. Some of these residues affect activity on all promoters and thus, are implicated in maintaining MarA structure whereas several solvent-exposed amino acids not involved in DNA binding were important for MarA activity on specific promoters. The pattern of activation defects defined a class II promoter-specific activating region. However, a localized class I activating region was not apparent. These results suggest that MarA activates transcription by at least two distinct mechanisms. Furthermore, the important role of phosphate contacts in marbox affinity suggests that indirect readout contributes to binding site recognition by MarA.

Promoter discrimination by the related transcriptional activators MarA and SoxS: differential regulation by differential binding.

MarA and SoxS are closely related proteins ( approximately 45% identical) that transcriptionally activate a common set of unlinked genes, resulting in multiple antibiotic and superoxide resistance in Escherichia coli. Both proteins bind as monomers to a 20 bp degenerate asymmetric recognition sequence, the 'marbox', located upstream of the promoter. However, the proteins differ widely in the extents to which they activate particular promoters, with the consequence that overexpression of SoxS leads to greater superoxide resistance than does overexpression of MarA. This 'discrimination' between activators by promoters was demonstrated in vivo, using promoters fused to lacZ, and in vitro, using purified RNA polymerase, promoter DNA and MarA or SoxS. The marbox was found to be a critical element in discrimination by in vivo and in vitro assays of hybrid promoters containing the marbox from one gene and the core promoter from another. Furthermore, by sequential mutation of its marbox, a promoter that discriminated 35-fold in favour of SoxS was converted into one that did not discriminate. The relative activation of a promoter by MarA or SoxS was paralleled by the relative binding of the two activators to the promoter's marbox as assayed by band shift experiments. Thus, differential recognition of closely related marbox sequences by the closely related activators is the primary basis for promoter discrimination. Discrimination enables the cell to customize its response to the stresses that trigger synthesis of the activators.

Attitudes of Omani physicians to people with epilepsy.

OBJECTIVE: This study reports the results of a questionnaire design to elicit doctors views about epilepsy. METHODS: Sixty-two percent of medical staff working in different regions of Oman responded. The questionnaire covered certain topics regarding the source of the knowledge of the doctors on seizure disorders and the personalities and behavior of people with epilepsy. RESULTS: The study suggests that although doctors in Oman gained knowledge on epilepsy prior to medical education, more doctors judged people with epilepsy `negatively` compared to `positively` for normal people. CONCLUSION: A developing country such as Oman needs to inculcate perceptions and attitude that are more realistic amongst their doctors toward people with epilepsy.

Structural requirements for marbox function in transcriptional activation of mar/sox/rob regulon promoters in Escherichia coli: sequence, orientation and spatial relationship to the core promoter.

The promoters of the mar/sox/rob regulon of Escherichia coli contain a binding site (marbox) for the homologous transcriptional activators MarA, SoxS and Rob. In spite of data from footprinting studies, the marbox has not been precisely defined because of its degeneracy and asymmetry and seemingly variable location with respect to the -10 and -35 hexamers for RNA polymerase (RNP) binding. Here, we use DNA retardation studies and hybrid promoters to identify optimally binding 20 bp minimal marboxes from a number of promoters. This has yielded a more defined marbox consensus sequence (AYnGCACnnWnnRYYAAAYn) and has led to the demonstration that some marboxes are inverted relative to others. Using transcriptional fusions to lacZ, we have found that only one marbox orientation is functional at a given location. Moreover, the functional orientation is determined by marbox location: marboxes that are 15 or more basepairs upstream of the -35 hexamer are oriented opposite those closer to the -35 hexamer. Marbox orientation and the spacing between marbox and signals for RNP binding are critical for transcriptional activation, presumably to align MarA with RNP.

Posterior chamber phakic intraocular lens for hyperopia.

PURPOSE: A Phase I U.S. FDA clinical study of a plate haptic posterior chamber phakic intraocular lens (STAAR Surgical Implantable Contact Lens) for treatment of hyperopia was conducted at 4 sites in the United States. The purpose of this report is to assess the short-term safety and efficacy. METHODS: Ten patients with hyperopia between +2.50 and +10.875 D were implanted in one eye each with the posterior chamber plate phakic intraocular lens and were examined at baseline and 1 day, 1 week, 1, 3, and 6 months after surgery. Mean baseline hyperopia was +6.63 D. RESULTS: At 6 months postoperatively, 7 of 10 eyes (70%) had an uncorrected visual acuity of 20/20 or better and 10 of 10 (100%) had 20/40 or better. Eight of ten eyes (80%) had a spectacle-corrected visual acuity within 1 line of baseline; the other two eyes (20%) had an improvement of 3 lines. Mean 6-month postoperative spherical equivalent refraction was +0.20 +/- 0.61D (range, -0.50 to +1.50 D), a reduction of 6.025 D from baseline. Eight of 10 eyes (80%) were within +/-0.50 D of emmetropia, 9 eyes (90%) were within +/-1.00 D, and all eyes (100%) were within +/-1.50 D. No operative or postoperative complications or adverse reactions were observed. CONCLUSIONS: Results support the short-term safety, efficacy, and predictability of the STAAR Surgical Implantable Contact Lens (plate haptic posterior chamber phakic intraocular lens) in the treatment of hyperopia.

A novel DNA-binding motif in MarA: the first structure for an AraC family transcriptional activator.

A crystal structure for a member of the AraC prokaryotic transcriptional activator family, MarA, in complex with its cognate DNA-binding site is described. MarA consists of two similar subdomains, each containing a helix-turn-helix DNA-binding motif. The two recognition helices of the motifs are inserted into adjacent major groove segments on the same face of the DNA but are separated by only 27 A thereby bending the DNA by approximately 35 degrees. Extensive interactions between the recognition helices and the DNA major groove provide the sequence specificity.