Redox-dependent condensation and cytoplasmic granulation by human ssDNA-binding protein-1 delineate roles in oxidative stress response.

Human single-stranded DNA binding protein 1 (hSSB1/NABP2/OBFC2B) plays central roles in DNA repair. Here, we show that purified hSSB1 undergoes redox-dependent liquid-liquid phase separation (LLPS) in the presence of single-stranded DNA or RNA, features that are distinct from those of LLPS by bacterial SSB. hSSB1 nucleoprotein droplets form under physiological ionic conditions in response to treatment modeling cellular oxidative stress. hSSB1's intrinsically disordered region is indispensable for LLPS, whereas all three cysteine residues of the oligonucleotide/oligosaccharide-binding fold are necessary to maintain redox-sensitive droplet formation. Proteins interacting with hSSB1 show selective enrichment inside hSSB1 droplets, suggesting tight content control and recruitment functions for the condensates. While these features appear instrumental for genome repair, we detected cytoplasmic hSSB1 condensates in various cell lines colocalizing with stress granules upon oxidative stress, implying extranuclear function in cellular stress response. Our results suggest condensation-linked roles for hSSB1, linking genome repair and cytoplasmic defense.

Highly sensitive mapping of in vitro type II topoisomerase DNA cleavage sites with SHAN-seq.

Type II topoisomerases (topos) are a ubiquitous and essential class of enzymes that form transient enzyme-bound double-stranded breaks on DNA called cleavage complexes. The location and frequency of these cleavage complexes on DNA is important for cellular function, genomic stability and a number of clinically important anticancer and antibacterial drugs, e.g. quinolones. We developed a simple high-accuracy end-sequencing (SHAN-seq) method to sensitively map type II topo cleavage complexes on DNA in vitro. Using SHAN-seq, we detected Escherichia coli gyrase and topoisomerase IV cleavage complexes at hundreds of sites on supercoiled pBR322 DNA, approximately one site every ten bp, with frequencies that varied by two-to-three orders of magnitude. These sites included previously identified sites and 20-50-fold more new sites. We show that the location and frequency of cleavage complexes at these sites are enzyme-specific and vary substantially in the presence of the quinolone, ciprofloxacin, but not with DNA supercoil chirality, i.e. negative versus positive supercoiling. SHAN-seq's exquisite sensitivity provides an unprecedented single-nucleotide resolution view of the distribution of gyrase and topoisomerase IV cleavage complexes on DNA. Moreover, the discovery that these enzymes can cleave DNA at orders of magnitude more sites than the relatively few previously known sites resolves the apparent paradox of how these enzymes resolve topological problems throughout the genome.

Highly sensitive mapping of in vitro type II topoisomerase DNA cleavage sites with SHAN-seq.

Type II topoisomerases (topos) are a ubiquitous and essential class of enzymes that form transient enzyme-bound double-stranded breaks on DNA called cleavage complexes. The location and frequency of these cleavage complexes on DNA is important for cellular function, genomic stability, and a number of clinically important anticancer and antibacterial drugs, e.g., quinolones. We developed a simple high-accuracy end-sequencing (SHAN-seq) method to sensitively map type II topo cleavage complexes on DNA in vitro. Using SHAN-seq, we detected Escherichia coli gyrase and topoisomerase IV cleavage complexes at hundreds of sites on supercoiled pBR322 DNA, approximately one site every ten bp, with frequencies that varied by two-to-three orders of magnitude. These sites included previously identified sites and 20-50 fold more new sites. We show that the location and frequency of cleavage complexes at these sites are enzyme-specific and vary substantially in the presence of the quinolone, ciprofloxacin, but not with DNA supercoil chirality, i.e., negative vs. positive supercoiling. SHAN-seq's exquisite sensitivity provides an unprecedented single-nucleotide resolution view of the distribution of gyrase and topoisomerase IV cleavage complexes on DNA. Moreover, the discovery that these enzymes can cleave DNA at orders of magnitude more sites than the relatively few previously known sites resolves the apparent paradox of how these enzymes resolve topological problems throughout the genome.

Bloom syndrome helicase contributes to germ line development and longevity in zebrafish.

RecQ helicases-also known as the "guardians of the genome"-play crucial roles in genome integrity maintenance through their involvement in various DNA metabolic pathways. Aside from being conserved from bacteria to vertebrates, their importance is also reflected in the fact that in humans impaired function of multiple RecQ helicase orthologs are known to cause severe sets of problems, including Bloom, Werner, or Rothmund-Thomson syndromes. Our aim was to create and characterize a zebrafish (Danio rerio) disease model for Bloom syndrome, a recessive autosomal disorder. In humans, this syndrome is characterized by short stature, skin rashes, reduced fertility, increased risk of carcinogenesis, and shortened life expectancy brought on by genomic instability. We show that zebrafish blm mutants recapitulate major hallmarks of the human disease, such as shortened lifespan and reduced fertility. Moreover, similarly to other factors involved in DNA repair, some functions of zebrafish Blm bear additional importance in germ line development, and consequently in sex differentiation. Unlike fanc genes and rad51, however, blm appears to affect its function independent of tp53. Therefore, our model will be a valuable tool for further understanding the developmental and molecular attributes of this rare disease, along with providing novel insights into the role of genome maintenance proteins in somatic DNA repair and fertility.

Evaluation and modulation of DNA lesion bypass in an SV40 large T antigen-based in vitro replication system.

DNA damage removal by nucleotide excision repair (NER) and replicative bypass via translesion synthesis (TLS) and template switch (TSw) are important in ensuring genome stability. In this study, we tested the applicability of an SV40 large T antigen-based replication system for the simultaneous examination of these damage tolerance processes. Using both Sanger and next-generation sequencing combined with lesion-specific qPCR and replication efficiency studies, we demonstrate that this system works well for studying NER and TLS, especially its one-polymerase branch, while it is less suited to investigations of homology-related repair processes, such as TSw. Cis-syn cyclobutane pyrimidine dimer photoproducts were replicated with equal efficiency to lesion-free plasmids in vitro, and the majority of TLS on this lesion could be inhibited by a peptide (PIR) specific for the polη-PCNA interaction interface. TLS on 6-4 pyrimidine-pyrimidone photoproduct proved to be inefficient and was slightly facilitated by PIR as well as by a recombinant ubiquitin-binding zinc finger domain of polη in HeLa extract, possibly by promoting polymerase exchange. Supplementation of the extract with recombinant PCNA variants indicated the dependence of TLS on PCNA ubiquitylation. In contrast to active TLS and NER, we found no evidence of successful TSw in cellular extracts. The established methods can promote in vitro investigations of replicative DNA damage bypass.

Human RAD51 rapidly forms intrinsically dynamic nucleoprotein filaments modulated by nucleotide binding state.

Formation of RAD51 filaments on single-stranded DNA is an essential event during homologous recombination, which is required for homology search, strand exchange and protection of replication forks. Formation of nucleoprotein filaments (NF) is required for development and genomic stability, and its failure is associated with developmental abnormalities and tumorigenesis. Here we describe the structure of the human RAD51 NFs and of its Walker box mutants using electron microscopy. Wild-type RAD51 filaments adopt an 'open' conformation when compared to a 'closed' structure formed by mutants, reflecting alterations in helical pitch. The kinetics of formation/disassembly of RAD51 filaments show rapid and high ssDNA coverage via low cooperativity binding of RAD51 units along the DNA. Subsequently, a series of isomerization or dissociation events mediated by nucleotide binding state creates intrinsically dynamic RAD51 NFs. Our findings highlight important a mechanistic divergence among recombinases from different organisms, in line with the diversity of biological mechanisms of HR initiation and quality control. These data reveal unexpected intrinsic dynamic properties of the RAD51 filament during assembly/disassembly, which may be important for the proper control of homologous recombination.

Recent adaptations of fluorescence techniques for the determination of mechanistic parameters of helicases and translocases.

Helicases and translocases are nucleic acid (NA)-based molecular motors that use the free energy liberated during the nucleoside triphosphate (NTP, usually ATP) hydrolysis cycle for unidirectional translocation along their NA (DNA, RNA or heteroduplex) substrates. Determination of the kinetic and thermodynamic parameters of their mechanoenzymatic cycle serves as a basis for the exploration of their physiological behavior and various cellular functions. Here we describe how recent adaptations of fluorescence-based solution kinetic methods can be used to determine practically all important mechanistic parameters of NA-based motor proteins. We outline practically useful analysis procedures for equilibrium, steady-state and transient kinetic data. This analysis can be used to quantitatively characterize the enzymatic steps of the NTP hydrolytic cycle, the binding site size, stoichiometry and energetics of protein-NA interactions, the rate and processivity of translocation along and unwinding of NA strands, and the mechanochemical coupling between these processes. The described methods yield insights into the functional role of the enzymes, and also greatly aid the design and interpretation of single-molecule experiments as well as the engineering of enzymatic properties for biotechnological applications.

A nucleotide-dependent and HRDC domain-dependent structural transition in DNA-bound RecQ helicase.

The allosteric communication between the ATP- and DNA-binding sites of RecQ helicases enables efficient coupling of ATP hydrolysis to translocation along single-stranded DNA (ssDNA) and, in turn, the restructuring of multistranded DNA substrates during genome maintenance processes. In this study, we used the tryptophan fluorescence signal of Escherichia coli RecQ helicase to decipher the kinetic mechanism of the interaction of the enzyme with ssDNA. Rapid kinetic experiments revealed that ssDNA binding occurs in a two-step mechanism in which the initial binding step is followed by a structural transition of the DNA-bound helicase. We found that the nucleotide state of RecQ greatly influences the kinetics of the detected structural transition, which leads to a high affinity DNA-clamped state in the presence of the nucleotide analog ADP-AlF4. The DNA binding mechanism is largely independent of ssDNA length, indicating the independent binding of RecQ molecules to ssDNA and the lack of significant DNA end effects. The structural transition of DNA-bound RecQ was not detected when the ssDNA binding capability of the helicase-RNase D C-terminal domain was abolished or the domain was deleted. The results shed light on the nature of conformational changes leading to processive ssDNA translocation and multistranded DNA processing by RecQ helicases.

A subdomain interaction at the base of the lever allosterically tunes the mechanochemical mechanism of myosin 5a.

The motor domain of myosin is the core element performing mechanochemical energy transduction. This domain contains the actin and ATP binding sites and the base of the force-transducing lever. Coordinated subdomain movements within the motor are essential in linking the ATPase chemical cycle to translocation along actin filaments. A dynamic subdomain interface located at the base of the lever was previously shown to exert an allosteric influence on ATP hydrolysis in the non-processive myosin 2 motor. By solution kinetic, spectroscopic and ensemble and single-molecule motility experiments, we determined the role of a class-specific adaptation of this interface in the mechanochemical mechanism of myosin 5a, a processive intracellular transporter. We found that the introduction of a myosin 2-specific repulsive interaction into myosin 5a via the I67K mutation perturbs the strong-binding interaction of myosin 5a with actin, influences the mechanism of ATP binding and facilitates ATP hydrolysis. At the same time, the mutation abolishes the actin-induced activation of ADP release and, in turn, slows down processive motility, especially when myosin experiences mechanical drag exerted by the action of multiple motor molecules bound to the same actin filament. The results highlight that subtle structural adaptations of the common structural scaffold of the myosin motor enable specific allosteric tuning of motor activity shaped by widely differing physiological demands.