Unwinding Rate Research Articles

Variable Inhibition of DNA Unwinding Rates Catalyzed by the SARS-CoV-2 Helicase Nsp13 by Structurally Distinct Single DNA Lesions.

The SARS-CoV-2 helicase, non-structural protein 13 (Nsp13), plays an essential role in viral replication, translocating in the 5' → 3' direction as it unwinds double-stranded RNA/DNA. We investigated the impact of structurally distinct DNA lesions on DNA unwinding catalyzed by Nsp13. The selected lesions include two benzo[a]pyrene (B[a]P)-derived dG adducts, the UV-induced cyclobutane pyrimidine dimer (CPD), and the pyrimidine (6-4) pyrimidone (6-4PP) photolesion. The experimentally observed unwinding rate constants (kobs) and processivities (P) were examined. Relative to undamaged DNA, the kobs values were diminished by factors of up to ~15 for B[a]P adducts but only by factors of ~2-5 for photolesions. A minor-groove-oriented B[a]P adduct showed the smallest impact on P, which decreased by ~11% compared to unmodified DNA, while an intercalated one reduced P by ~67%. However, the photolesions showed a greater impact on the processivities; notably, the CPD, with the highest kobs value, exhibited the lowest P, which was reduced by ~90%. Our findings thus show that DNA unwinding efficiencies are lesion-dependent and most strongly inhibited by the CPD, leading to the conclusion that processivity is a better measure of DNA lesions' inhibitory effects than unwinding rate constants.

Dysregulated DnaB unwinding induces replisome decoupling and daughter strand gaps that are countered by RecA polymerization.

The replicative helicase, DnaB, is a central component of the replisome and unwinds duplex DNA coupled with immediate template-dependent DNA synthesis by the polymerase, Pol III. The rate of helicase unwinding is dynamically regulated through structural transitions in the DnaB hexamer between dilated and constricted states. Site-specific mutations in DnaB enforce a faster more constricted conformation that dysregulates unwinding dynamics, causing replisome decoupling that generates excess ssDNA and induces severe cellular stress. This surplus ssDNA can stimulate RecA recruitment to initiate recombinational repair, restart, or activation of the transcriptional SOS response. To better understand the consequences of dysregulated unwinding, we combined targeted genomic dnaB mutations with an inducible RecA filament inhibition strategy to examine the dependencies on RecA in mitigating replisome decoupling phenotypes. Without RecA filamentation, dnaB:mut strains had reduced growth rates, decreased mutagenesis, but a greater burden from endogenous damage. Interestingly, disruption of RecA filamentation in these dnaB:mut strains also reduced cellular filamentation but increased markers of double strand breaks and ssDNA gaps as detected by in situ fluorescence microscopy and FACS assays, TUNEL and PLUG, respectively. Overall, RecA plays a critical role in strain survival by protecting and processing ssDNA gaps caused by dysregulated helicase activity in vivo.

Characterization of human XPD helicase activity with single-molecule magnetic tweezers

XPD helicase is a DNA-unwinding enzyme involved in DNA repair. As part of TFIIH, XPD opens a repair bubble in DNA for access by proteins in the nucleotide excision repair pathway. XPD uses the energy from ATP hydrolysis to translocate in the 5′ to 3′ direction on one strand of duplex DNA, displacing the opposite strand in the process. We used magnetic tweezers assays to measure the double-stranded DNA unwinding and single-stranded DNA translocation activities of human XPD in isolation. In our experimental setup, hXPD exhibited low unwinding processivity of ∼14 bp and slow unwinding rate of ∼0.3 bp/s. Measurements of the ssDNA translocation activity demonstrated that hXPD translocated on ssDNA at a similar rate as unwinding, revealing that slow rate was an intrinsic property of the hXPD translocation. Individual unwinding and translocation events were composed of pauses and runs with a distribution of lengths and rates. Analysis of these events unveiled similar mean run lengths and rates for unwinding and translocation, indicating that the unwinding behavior was a direct reflection of the translocation activity. The analysis also revealed that hXPD spent similar time stalling and unwinding/translocating. The detailed basal activity of hXPD reported here provides a baseline for future studies on how hXPD activity is regulated by other TFIIH components.

E. Coli RecB Nuclease Domain Regulates RecBCD Helicase Activity but not Single Stranded DNA Translocase Activity

Much is still unknown about the mechanisms by which helicases unwind duplex DNA. Whereas structure-based models describe DNA unwinding as occurring by the ATPase motors mechanically pulling the DNA duplex across a wedge domain in the helicase, biochemical data show that processive DNA unwinding by E. coli RecBCD helicase can occur in the absence of ssDNA translocation by the canonical RecB and RecD motors. Here we show that DNA unwinding is not a simple consequence of ssDNA translocation by the motors. Using stopped-flow fluorescence approaches, we show that a RecB nuclease domain deletion variant (RecBΔNucCD) unwinds dsDNA at significantly slower rates than RecBCD, while the ssDNA translocation rate is unaffected. This effect is primarily due to the absence of the nuclease domain since a nuclease-dead mutant (RecBD1080ACD), which retains the nuclease domain, showed no change ssDNA translocation or dsDNA unwinding rates relative to RecBCD on short DNA substrates (≤60 base pairs). Hence, ssDNA translocation is not rate-limiting for DNA unwinding. RecBΔNucCD also initiates unwinding much slower than RecBCD from a blunt-ended DNA. RecBΔNucCD also unwinds DNA ∼two-fold slower than RecBCD on long DNA (∼20 kilo base pair) in single molecule optical tweezer experiments, although the rates for RecBD1080ACD unwinding are intermediate between RecBCD and RecBΔNucCD. Surprisingly, significant pauses in DNA unwinding occur even in the absence of chi (crossover hotspot instigator) sites. We hypothesize that the nuclease domain influences the rate of DNA base pair melting, possibly allosterically and that RecBΔNucCD may mimic a post-chi state of RecBCD.

Molecular wrench activity of DNA helicases: Keys to modulation of rapid kinetics in DNA repair.

DNA helicase activity is essential for the vital DNA metabolic processes of recombination, replication, transcription, translation, and repair. Recently, an unexpected, rapid exponential ATP-stimulated DNA unwinding rate was observed from an Archaeoglobus fulgidus helicase (AfXPB) as compared to the slower conventional helicases from Sulfolobus tokodaii, StXPB1 and StXPB2. This unusual rapid activity suggests a "molecular wrench" mechanism arising from the torque applied by AfXPB on the duplex structure in transitioning from open to closed conformations. However, much remains to be understood. Here, we investigate the concentration dependence of DNA helicase binding and ATP-stimulated kinetics of StXPB2 and AfXPB, as well as their binding and activity in Bax1 complexes, via an electrochemical assay with redox-active DNA monolayers. StXPB2 ATP-stimulated activity is concentration-independent from 8 to 200 nM. Unexpectedly, AfXPB activity is concentration-dependent in this range, with exponential rate constants varying from seconds at concentrations greater than 20 nM to thousands of seconds at lower concentrations. At 20 nM, rapid exponential signal decay ensues, linearly reverses, and resumes with a slower exponential decay. This change in AfXPB activity as a function of its concentration is rationalized as the crossover between the fast molecular wrench and slower conventional helicase modes. AfXPB-Bax1 inhibits rapid activity, whereas the StXPB2-Bax1 complex induces rapid kinetics at higher concentrations. This activity is rationalized with the crystal structures of these complexes. These findings illuminate the different physical models governing molecular wrench activity for improved biological insight into a key factor in DNA repair.

Human XPD is an active helicase with low processivity and overall unwinding rate.

XPD helicase is a DNA unwinding enzyme involved in multiple cellular processes. Of particular note is its role in the large multidomain complex transcription factor IIH (TFIIH). As part of TFIIH, XPD opens a bubble of ∼ 26 base pairs of DNA for access by proteins in the nucleotide excision repair pathway (NER). XPD uses the energy from ATP to translocate in the 5’ to 3’ direction on one strand of duplex DNA, displacing the opposite strand in the process. We have measured the double-stranded DNA (dsDNA) unwinding and single-stranded DNA (ssDNA) translocation activities of human XPD using magnetic tweezers assays. By following changes in the extension of tethered DNA molecules under force, we are able to measure the movement of individual helicase molecule on DNA in real time. Processivity, overall rate, pause position, step size, and step rates for both dsDNA unwinding and ssDNA translocation of XPD are measured and compared. The enzyme exhibits low processivity in helicase activity, with an average ∼10 bases unwound, while the translocation of the enzyme on ssDNA is more processive, with ∼30 bases translocated. This difference in processivity on dsDNA unwinding vs ssDNA translocation may be due to interactions between the displaced strand and XPD limiting the overall processivity. However, these interactions do not affect rates, as same average rate (∼0.3 nucleotides/s) for both unwinding and ssDNA translocation has been observed. This same rate also indicates that the enzyme works as an active helicase. There is no obvious correlation between pause positions and DNA sequence. The observed low unwinding processivity (lower than the expected NER repair bubble) and rate suggests that other proteins in the TFIIH complex enhance the helicase activity of XPD.

UPF1 mutants with intact ATPase but deficient helicase activities promote efficient nonsense-mediated mRNA decay.

The conserved RNA helicase UPF1 coordinates nonsense-mediated mRNA decay (NMD) by engaging with mRNAs, RNA decay machinery, and the terminating ribosome. UPF1 ATPase activity is implicated in mRNA target discrimination and completion of decay, but the mechanisms through which UPF1 enzymatic activities such as helicase, translocase, RNP remodeling, and ATPase-stimulated dissociation influence NMD remain poorly defined. Using high-throughput biochemical assays to quantify UPF1 enzymatic activities, we show that UPF1 is only moderately processive (<200 nt) in physiological contexts and undergoes ATPase-stimulated dissociation from RNA. We combine an in silico screen with these assays to identify and characterize known and novel UPF1 mutants with altered helicase, ATPase, and RNA binding properties. We find that UPF1 mutants with substantially impaired processivity, slower unwinding rate, and/or reduced mechanochemical coupling (i.e. the ability to harness ATP hydrolysis for work) still support efficient NMD in human cells. These data are consistent with a central role for UPF1 ATPase activity in driving cycles of RNA binding and dissociation to ensure accurate NMD target selection.

Inhibition of E. coli RecQ Helicase Activity by Structurally Distinct DNA Lesions: Structure—Function Relationships

DNA helicase unwinding activity can be inhibited by small molecules and by covalently bound DNA lesions. Little is known about the relationships between the structural features of DNA lesions and their impact on unwinding rates and processivities. Employing E.coli RecQ helicase as a model system, and various conformationally defined DNA lesions, the unwinding rate constants kobs = kU + kD, and processivities P = (kU/(kU + kD) were determined (kU, unwinding rate constant; kD, helicase-DNA dissociation rate constant). The highest kobs values were observed in the case of intercalated benzo[a]pyrene (BP)-derived adenine adducts, while kobs values of guanine adducts with minor groove or base-displaced intercalated adduct conformations were ~10-20 times smaller. Full unwinding was observed in each case with the processivity P = 1.0 (100% unwinding). The kobs values of the non-bulky lesions T(6-4)T, CPD cyclobutane thymine dimers, and a guanine oxidation product, spiroiminodihydantoin (Sp), are up to 20 times greater than some of the bulky adduct values; their unwinding efficiencies are strongly inhibited with processivities P = 0.11 (CPD), 0.062 (T(6-4)T), and 0.63 (Sp). These latter observations can be accounted for by correlated decreases in unwinding rate constants and enhancements in the helicase DNA complex dissociation rate constants.

UPF1 mutants with intact ATPase but deficient helicase activities promote efficient nonsense-mediated mRNA decay.

The conserved RNA helicase UPF1 coordinates nonsense-mediated mRNA decay (NMD) by engaging with mRNAs, RNA decay machineryand the terminating ribosome. UPF1 ATPase activity is implicated in mRNA target discrimination and completion of decay, but the mechanisms through which UPF1 enzymatic activities such as helicase, translocase, RNP remodeling, and ATPase-stimulated dissociation influence NMD remain poorly defined. Using high-throughput biochemical assays to quantify UPF1 enzymatic activities, we show that UPF1 is only moderately processive (<200 nt) in physiological contexts and undergoes ATPase-stimulated dissociation from RNA. We combine an in silico screen with these assays to identify and characterize known and novel UPF1 mutants with altered helicase, ATPase, and RNA binding properties. We find that UPF1 mutants with substantially impaired processivity (E797R, G619K/A546H), faster (G619K) or slower (K547P, E797R, G619K/A546H) unwinding rates, and/or reduced mechanochemical coupling (i.e. the ability to harness ATP hydrolysis for work;K547P, R549S, G619K, G619K/A546H) can still support efficient NMD of well-characterized targets in human cells. These data are consistent with a central role for UPF1 ATPase activity in driving cycles of RNA binding and dissociation to ensure accurate NMD target selection.

Auxiliary ATP binding sites support DNA unwinding by RecBCD

The RecBCD helicase initiates double-stranded break repair in bacteria by processively unwinding DNA with a rate approaching ∼1,600 bp·s−1, but the mechanism enabling such a fast rate is unknown. Employing a wide range of methodologies — including equilibrium and time-resolved binding experiments, ensemble and single-molecule unwinding assays, and crosslinking followed by mass spectrometry — we reveal the existence of auxiliary binding sites in the RecC subunit, where ATP binds with lower affinity and distinct chemical interactions as compared to the known catalytic sites. The essentiality and functionality of these sites are demonstrated by their impact on the survival of E.coli after exposure to damage-inducing radiation. We propose a model by which RecBCD achieves its optimized unwinding rate, even when ATP is scarce, by using the auxiliary binding sites to increase the flux of ATP to its catalytic sites.

Alignment of helicases on single-stranded DNA increases activity.

Helicases function in most biological processes that utilize RNA or DNA nucleic acids including replication, recombination, repair, transcription, splicing, and translation. They are motor proteins that bind ATP and then catalyze hydrolysis to release energy which is transduced for conformational changes. Different conformations correspond to different steps in a process that results in movement of the enzyme along the nucleic acid track in a unidirectional manner. Some helicases such as DEAD-box helicases do not translocate, but these enzymes transduce chemical energy from ATP hydrolysis to unwind secondary structure in DNA or RNA. Some helicases function as monomers while others assemble into defined structures, either dimers or higher order oligomers. Dda helicase from bacteriophage T4 and NS3 helicase domain from the hepatitis C virus are examples of monomeric helicases. These helicases can bind to single-stranded DNA in a manner that appears like train engines on a track. When monomeric helicases align on DNA, the activity of the enzymes increases. Helicase activity can include the rate of duplex unwinding and the total number of base pairs melted during a single binding event or processivity. Dda and NS3h are considered as having low processivity, unwinding fewer than 50 base pairs per binding event. Here, we report fusing two molecules of NS3h molecules together through genetically linking the C-terminus of one molecule to the N-terminus of a second NS3h molecule. We observed increased processivity relative to NS3h possibly arising from the increased probability that at least one of the helicases will completely unwind the DNA prior to dissociation. The dimeric enzyme also binds DNA more like the full-length NS3 helicase. Finally, the dimer can displace streptavidin from biotin-labeled oligonucleotide, whereas monomeric NS3h cannot.

Features of changes in the level of fibrosis markers in lipotoxic myocardial damage in patients with epicardial obesity

Abstract In visceral obesity, lipotoxic myocardial damage develops, which leads to the formation of myocardial fibrosis with impaired diastolic function and the development of heart failure (HF). Currently, the search for markers of early diagnosis of left ventricular (LV) diastolic dysfunction (DD) is relevant. Objective To study the features of changes in the level of fibrosis markers in lipotoxic myocardial damage in patients with epicardial obesity (EO). Materials and methods The study included 125 men with general obesity (BMI average 33.24±3.65 kg / sq. m.). All patients underwent transthoracic echocardiography (ECG) to assess the thickness of epicardial adipose tissue (tEAT) as the equivalent of visceral obesity, as well as to diagnose DD LV. According to the results of echocardiography, the patients were divided into 2 groups: EO(+) with epicardial fat thickness (tEAT) ≥7 mm (n=78); EO(−) with tEAT &amp;lt;7 mm (n=40). DD LV was detected in 7 patients, who were subsequently excluded from the analysis. All patients were assessed for profibrotic markers in the blood serum (MMP-3, collagen I, collagen III, TGF – β, VEGFA, PICP) using enzyme immunoassay. The level of free fatty acids (FFA), as early markers of lipotoxic myocardial damage, was also determined in all patients using enzymelinked immunosorbent assay. The peak untwist ratio LV was studied using speckle-tracking ECG. The exclusive criteria were the presence of coronary pathology, arterial hypertension, type 2 diabetes mellitus, as possible causes of lipotoxic myocardial damage. Results When studying the features of changes in the level of serum markers of myocardial fibrosis, a statistically significant increase in the level of all the studied markers was revealed in the EO(+) group compared to the EO(−) group, and a statistically significant increase in the level of FFA in the EO(+) group compared to the EO(−) group (0.84±0.02 mmol/l and 0.35±0.01 mmol/L, respectively, p=0.02). A statistically significant relationship was found between the levels of FFA and collagen III (r=0.32, p=0.03) and TGF – β (r=0.30, p=0.04). According to the results of speckle-tracking ECG in the EO(+) group, an increase in the rate of LV untwist to −125.56 (−141.0; −117.0) deg/s (p=0.003) was determined. When studying the effect of FFA as a marker of lipotoxic effect on the myocardium, a weak statistically significant relationship was revealed between the level of FFA and the rate of LV untwist (r=0.27; p=0.03) in the EO(+) group, while no such effect was found in the EO(−) group. Conclusion In EO, as a result of neurohumoral disorders, lipotoxic myocardial damage develops and progresses, leading to LV DD, the detection of which is probably possible with the help of the LV unwinding rate. Funding Acknowledgement Type of funding sources: None.

Rad53 checkpoint kinase regulation of DNA replication fork rate via Mrc1 phosphorylation.

The Rad53 DNA checkpoint protein kinase plays multiple roles in the budding yeast cell response to DNA replication stress. Key amongst these is its enigmatic role in safeguarding DNA replication forks. Using DNA replication reactions reconstituted with purified proteins, we show Rad53 phosphorylation of Sld3/7 or Dbf4-dependent kinase blocks replication initiation whilst phosphorylation of Mrc1 or Mcm10 slows elongation. Mrc1 phosphorylation is necessary and sufficient to slow replication forks in complete reactions; Mcm10 phosphorylation can also slow replication forks, but only in the absence of unphosphorylated Mrc1. Mrc1 stimulates the unwinding rate of the replicative helicase, CMG, and Rad53 phosphorylation of Mrc1 prevents this. We show that a phosphorylation-mimicking Mrc1 mutant cannot stimulate replication in vitro and partially rescues the sensitivity of a rad53 null mutant to genotoxic stress in vivo. Our results show that Rad53 protects replication forks in part by antagonising Mrc1 stimulation of CMG unwinding.

The helicase core accessory regions of the phage BFK20 DnaB-like helicase gp43 significantly affect its activity, oligomeric state and DNA binding properties

The multifunctional phage replication protein gp43 is composed of an N-terminal prim-pol domain and a C-terminal domain similar to the SF4-type replicative helicases. We prepared four mutants all missing the prim-pol domain with the helicase core flanked by accessory N- and C-terminal regions truncated to varying extents. The shortest fragment still possessing strong ssDNA-dependent ATPase activity and helicase activity was gp43HEL519-983. The other proteins tested were gp43HEL557-983, gp43HEL519-855 and gp43HEL519-896. Removal of the 38 N-terminal residues in gp43HEL557-983, or the 128 and 87 C-terminal residues in gp43HEL519-855 and gp43HEL519-896, resulted in a significant decrease in the ATPase activities. The 38-amino acid N-terminal region has probably a function in modulating DNA binding and protein oligomerization. Deletion of the 87 C-terminal residues resulted in a twofold increase in the unwinding rate. This region is likely indispensable for binding to DNA substrates.

Using Atomic Force Microscopy to Study the Real Time Dynamics of DNA Unwinding by Mitochondrial Twinkle Helicase.

Understanding the structure and dynamics of DNA-protein interactions during DNA replication is crucial for elucidating the origins of disorders arising from its dysfunction. In this study, we employed Atomic Force Microscopy as a single-molecule imaging tool to examine the mitochondrial DNA helicase Twinkle and its interactions with DNA. We used imaging in air and time-lapse imaging in liquids to observe the DNA binding and unwinding activities of Twinkle hexamers at the single-molecule level. These procedures helped us visualize Twinkle loading onto and unloading from the DNA in the open-ring conformation. Using traditional methods, it has been shown that Twinkle is capable of unwinding dsDNA up to 20-55 bps. We found that the addition of mitochondrial single-stranded DNA binding protein (mtSSB) facilitates a 5-fold increase in the DNA unwinding rate for the Twinkle helicase. The protocols developed in this study provide new platforms to examine DNA replication and to explore the mechanism driving DNA deletion and human diseases. Graphic abstract: Mitochondrial Twinkle Helicase Dynamics.

Duplex DNA engagement and RPA oppositely regulate the DNA-unwinding rate of CMG helicase

A ring-shaped helicase unwinds DNA during chromosome replication in all organisms. Replicative helicases generally unwind duplex DNA an order of magnitude slower compared to their in vivo replication fork rates. However, the origin of slow DNA unwinding rates by replicative helicases and the mechanism by which other replication components increase helicase speed are unclear. Here, we demonstrate that engagement of the eukaryotic CMG helicase with template DNA at the replication fork impairs its helicase activity, which is alleviated by binding of the single-stranded DNA binding protein, RPA, to the excluded DNA strand. Intriguingly, we found that, when stalled due to interaction with the parental duplex, DNA rezipping-induced helicase backtracking reestablishes productive helicase-fork engagement, underscoring the significance of plasticity in helicase action. Our work provides a mechanistic basis for relatively slow duplex unwinding by replicative helicases and explains how replisome components that interact with the excluded DNA strand stimulate fork rates.

Characterzing the Auxiliary Nucleotide Binding Sites in RecBCD DNA Helicase, Towards Deciphering their Functional Role

RecBCD is a DNA helicase-nuclease, powering the initiation of ds break repair. It is a highly processive fast helicase, with unwinding rates approaching 1,600 bp/s. By employing a combination of equilibrium and time-resolved unwinding and binding experiments(both ensemble and single-molecule) to unravel RecBCD's molecular mechanism, we had demonstrated the existence of auxiliary binding sites in RecBCD, specifically in the RecC subunit, where ATP binds with lower affinity and with distinct chemical interactions as compared to the known catalytic sites. According to our model, RecBCD achieves its fast unwinding rate by utilizing the auxiliary binding sites to increase the flux of ATP to its catalytic sites. The number of nucleotide-binding sites determined by equilibrium dialysis has been estimated to be at least four. While RecB and RecD, each have one known structurally and chemically well-defined nucleotide-binding site, the additional two (or more) auxiliary nucleotide-binding sites have not been located. Our current study strongly supports that they are likely to be located in RecC. In this work, we have attempted to abolish the nucleotide-binding sites in RecC by using a multi-step approach. A list of possible nucleotide-binding sites was obtained by using a combination of UV-crosslinking, mass spectrometry and molecular docking studies. Based on this, several mutations in RecC were carefully designed and successfully cloned and purified to produce RecBCmutD. RecBCmutD has an activated DNA ATPase activity, with a higher KM,ATP. Furthermore, equilibrium nucleotide binding assay suggests that the RecBCmutD does not display the same biphasic binding isotherm as the WT RecBCD. Based on this, we propose that the auxiliary binding sites play an integrated role in the reaction mechanism of RecBCD.

Ring-shaped replicative helicase encircles double-stranded DNA during unwinding.

Ring-shaped replicative helicases are hexameric and play a key role in cellular DNA replication. Despite their importance, our understanding of the unwinding mechanism of replicative helicases is far from perfect. Bovine papillomavirus E1 is one of the best-known model systems for replicative helicases. E1 is a multifunctional initiator that senses and melts the viral origin and unwinds DNA. Here, we study the unwinding mechanism of E1 at the single-molecule level using magnetic tweezers. The result reveals that E1 as a single hexamer is a poorly processive helicase with a low unwinding rate. Tension on the DNA strands impedes unwinding, indicating that the helicase interacts strongly with both DNA strands at the junction. While investigating the interaction at a high force (26–30 pN), we discovered that E1 encircles dsDNA. By comparing with the E1 construct without a DNA binding domain, we propose two possible encircling modes of E1 during active unwinding.

Homology sensing via non-linear amplification of sequence-dependent pausing by RecQ helicase.

RecQ helicases promote genomic stability through their unique ability to suppress illegitimate recombination and resolve recombination intermediates. These DNA structure-specific activities of RecQ helicases are mediated by the helicase-and-RNAseD like C-terminal (HRDC) domain, via unknown mechanisms. Here, employing single-molecule magnetic tweezers and rapid kinetic approaches we establish that the HRDC domain stabilizes intrinsic, sequence-dependent, pauses of the core helicase (lacking the HRDC) in a DNA geometry-dependent manner. We elucidate the core unwinding mechanism in which the unwinding rate depends on the stability of the duplex DNA leading to transient sequence-dependent pauses. We further demonstrate a non-linear amplification of these transient pauses by the controlled binding of the HRDC domain. The resulting DNA sequence- and geometry-dependent pausing may underlie a homology sensing mechanism that allows rapid disruption of unstable (illegitimate) and stabilization of stable (legitimate) DNA strand invasions, which suggests an intrinsic mechanism of recombination quality control by RecQ helicases.

Impairment of testicular function in electronic cigarette (e-cig, e-cigs) exposed rats under low-voltage and nicotine-free conditions

Despite the lack of knowledge of the effects of electronic cigarettes (e-cigarettes, e-cigs) on public health, they have been proposed as a part of smoking cessation efforts. Recently, several basic scientific studies have pointed out how e-cigs can generate carcinogens, such as e-cig liquid thermal degradation by-products, and how the exposure can lead to genomic damage through inhibiting DNA repair or disrupting the redox homeostasis. However, scientific studies have pointed out how e-cigs can generate carcinogens and their release could be avoided setting the device to a low-voltage regimen. To test this feasibility, we show the effects of e-cig vapour generated from a low-voltage device filled with a nicotine-free liquid on rat testicular functions. The chemical analysis revealed the presence of carbonyls, such as formaldehyde, acetaldehyde and acrolein. Rats exposed reported a lower relative testis weight and higher levels of lactate dehydrogenase (LDH) as tissue damage marker, along with an impairment of 3β-hydroxysteroid dehydrogenase (3β-HSD), 17β-hydroxysteroid dehydrogenase (17β-HSD) and glucose-6-phosphate dehydrogenase (G6PDH) as key enzymes in the steroidogenesis pathway. The pro-oxidative environment was confirmed by the higher amount of reactive oxygen species (ROS), the development of lipid peroxidation and protein carbonylation, as well as from the disruption of antioxidant capability. Finally, we observed a higher rate of DNA unwinding in white blood cell line and boosted lipoxygenase (LOX)-linked activity, a tumour promotion marker. Even with the device setting at weak conditions, our results if extrapolated to humans suggest that exposure to e-cig vapours might alter gonads function in male vapers.