Single stranded DNA translocation of E. coli UvrD monomer is tightly coupled to ATP hydrolysis

E. coli UvrD is an SF1A helicase/translocase that functions in several DNA repair pathways. A UvrD monomer is a rapid and processive single-stranded (ss) DNA translocase, but is unable to unwind DNA processively in vitro. Based on data at saturating ATP (500 μM) we proposed a non-uniform stepping mechanism in which a UvrD monomer translocates with biased (3′ to 5′) directionality while hydrolyzing 1 ATP per DNA base translocated, but with a kinetic step-size of 4–5 nucleotides/step, suggesting a pause occurs every 4–5 nucleotides translocated. To further test this mechanism we examined UvrD translocation over a range of lower ATP concentrations (10–500 μM ATP), using transient kinetic approaches. We find a constant ATP coupling stoichiometry of ~1 ATP/DNA base translocated even at the lowest ATP concentration examined (10 μM) indicating that ATP hydrolysis is tightly coupled to forward translocation of a UvrD monomer along ssDNA with little slippage or futile ATP hydrolysis during translocation. The translocation kinetic step size remains constant at 4–5 nucleotides/step down to 50 μM ATP, but increases to ~7 nucleotides/step at 10 μM ATP. These results suggest that UvrD pauses more frequently during translocation at low ATP, but with little futile ATP hydrolysis

Srs2 Disassembles Rad51 Filaments by a Protein-Protein Interaction Triggering ATP Turnover and Dissociation of Rad51 from DNA

Rad51 is a DNA recombinase functioning in the repair of DNA double-strand breaks and the generation of genetic diversity by homologous recombination (HR). In the presence of ATP, Rad51 self-assembles into an extended polymer on single-stranded DNA to catalyze strand exchange. Inappropriate HR causes genomic instability, and it is normally prevented by remodeling enzymes that antagonize the activities of Rad51 nucleoprotein filaments. In yeast, the Srs2 helicase/translocase suppresses HR by clearing Rad51 polymers from single-stranded DNA. We have examined the mechanism of disassembly of Rad51 nucleoprotein filaments by Srs2 and find that a physical interaction between Rad51 and the C-terminal region of Srs2 triggers ATP hydrolysis within the Rad51 filament, causing Rad51 to dissociate from DNA. This allosteric mechanism explains the biological specialization of Srs2 as a DNA motor protein that antagonizes HR

Ensemble Methods for Monitoring Enzyme Translocation along Single Stranded Nucleic Acids

We review transient kinetic methods developed to study the mechanism of translocation of nucleic acid motor proteins. One useful stopped-flow fluorescence method monitors arrival of the translocase at the end of a fluorescently labeled nucleic acid. When conducted under single-round conditions the time courses can be analyzed quantitatively using n-step sequential models to determine the kinetic parameters for translocation (rate, kinetic step size and processivity). The assay and analysis discussed here can be used to study enzyme translocation along a linear lattice such as ssDNA or ssRNA. We outline the methods for experimental design and two approaches, along with their limitations, that can be used to analyze the time courses. Analysis of the full time courses using n-step sequential models always yields an accurate estimate of the translocation rate. An alternative semi-quantitative “time to peak” analysis yields accurate estimates of translocation rates only if the enzyme initiates translocation from a unique site on the nucleic acid. However, if initiation occurs at random sites along the nucleic acid, then the “time to peak” analysis can yield inaccurate estimates of even the rates of translocation depending on the values of other kinetic parameters, especially the rate of dissociation of the translocase. Thus, in those cases analysis of the full time course is needed to obtain accurate estimates of translocation rates

Translocation of Saccharomyces cerevisiae Pif1 helicase monomers on single-stranded DNA

In Saccharomyces cerevisiae Pif1 participates in a wide variety of DNA metabolic pathways both in the nucleus and in mitochondria. The ability of Pif1 to hydrolyse ATP and catalyse unwinding of duplex nucleic acid is proposed to be at the core of its func-tions. We recently showed that upon binding to DNA Pif1 dimerizes and we proposed that a dimer of Pif1 might be the species poised to catalysed DNA un-winding. In this work we show that monomers of Pif1 are able to translocate on single-stranded DNA with 50 to 30 directionality. We provide evidence that the translocation activity of Pif1 could be used in activities other than unwinding, possibly to displace proteins from ssDNA. Moreover, we show that monomers of Pif1 retain some unwinding activity although a dimer is clearly a better helicase, suggesting that regulation of the oligomeric state of Pif1 could play a role in its functioning as a helicase or a translocase. Finally, although we show that Pif1 can translocate on ssDNA, the translocation profiles suggest the presence on ssDNA of two populations of Pif1, both able to translocate with 50 to 30 directionality

High-throughput, fluorescent-aptamer-based measurements of steady-state transcription rates for the Mycobacterium tuberculosis RNA polymerase

The first step in gene expression is the transcription of DNA sequences into RNA. Regulation at the level of transcription leads to changes in steady-state concentrations of RNA transcripts, affecting the flux of downstream functions and ultimately cellular phenotypes. Changes in transcript levels are routinely followed in cellular contexts via genome-wide sequencing techniques. However, in vitro mechanistic studies of transcription have lagged with respect to throughput. Here, we describe the use of a real-time, fluorescent-aptamer-based method to quantitate steady-state transcription rates of the Mycobacterium tuberculosis RNA polymerase. We present clear controls to show that the assay specifically reports on promoter-dependent, full-length RNA transcription rates that are in good agreement with the kinetics determined by gel-resolved, α-32P NTP incorporation experiments. We illustrate how the time-dependent changes in fluorescence can be used to measure regulatory effects of nucleotide concentrations and identity, RNAP and DNA concentrations, transcription factors, and antibiotics. Our data showcase the ability to easily perform hundreds of parallel steady-state measurements across varying conditions with high precision and reproducibility to facilitate the study of the molecular mechanisms of bacterial transcription

Correspondence Between Perceived Pubertal Development and Hormone Levels in 9-10 Year-Olds From the Adolescent Brain Cognitive Development Study.

Aim: To examine individual variability between perceived physical features and hormones of pubertal maturation in 9-10-year-old children as a function of sociodemographic characteristics. Methods: Cross-sectional metrics of puberty were utilized from the baseline assessment of the Adolescent Brain Cognitive Development (ABCD) Study—a multi-site sample of 9–10 year-olds (n = 11,875)—and included perceived physical features via the pubertal development scale (PDS) and child salivary hormone levels (dehydroepiandrosterone and testosterone in all, and estradiol in females). Multi-level models examined the relationships among sociodemographic measures, physical features, and hormone levels. A group factor analysis (GFA) was implemented to extract latent variables of pubertal maturation that integrated both measures of perceived physical features and hormone levels. Results: PDS summary scores indicated more males (70%) than females (31%) were prepubertal. Perceived physical features and hormone levels were significantly associated with child\u27s weight status and income, such that more mature scores were observed among children that were overweight/obese or from households with low-income. Results from the GFA identified two latent factors that described individual differences in pubertal maturation among both females and males, with factor 1 driven by higher hormone levels, and factor 2 driven by perceived physical maturation. The correspondence between latent factor 1 scores (hormones) and latent factor 2 scores (perceived physical maturation) revealed synchronous and asynchronous relationships between hormones and concomitant physical features in this large young adolescent sample. Conclusions: Sociodemographic measures were associated with both objective hormone and self-report physical measures of pubertal maturation in a large, diverse sample of 9-10 year-olds. The latent variables of pubertal maturation described a complex interplay between perceived physical changes and hormone levels that hallmark sexual maturation, which future studies can examine in relation to trajectories of brain maturation, risk/resilience to substance use, and other mental health outcomes

Transient-State Kinetic Studies of Escherichia coli UvrD Monomer Translocation along Single-Stranded DNA

E. coli UvrD is a DNA helicase that functions in a variety of DNA metabolic processes. I investigated the ssDNA translocation mechanism of the UvrD monomer, a required component of the dimeric UvrD helicase. UvrD is a superfamily I DNA helicase/ translocase that unwinds DNA and translocates along ssDNA, 3’ to 5’, using an ATPdependent mechanism. Using transient-state kinetic approaches I studied UvrD monomer translocation by monitoring three features of the translocation process as a function of ssDNA length: 1.) Arrival of the UvrD monomer at the 5’-end of the ssDNA; 2.) ATP hydrolysis during UvrD translocation; and 3.) UvrD dissociation from the ssDNA during translocation. Global analysis of all three time courses to an n-step sequential mechanism provides estimates of the translocation rate, processivity, kinetic step size, ATP coupling stoichiometry, and UvrD dissociation rate constants from the ssDNA, placing constraints on possible translocation mechanisms. I studied UvrD monomer translocation over a range of ATP and salt concentrations, in the presence of duplex DNA structures, and on different ssDNA base compositions to determine the UvrD monomer translocation mechanism and how it is affected by changes in the DNA substrate. I found that the UvrD monomer translocation mechanism along ssDNA is consistent with an inchworm stepping model where the UvrD monomer hydrolyzes one ATP per nucleotide translocated (10 mM Tris-HCl, pH 8.3, 20 mM NaCl, 20% (v/v) glycerol, and 2 mM MgCl2 at 25°C). This ATP coupling stoichiometry did not change over a range of ATP concentrations (10-500 µM) spanning above and below the Michaelis-Menten constant for ATP binding to UvrD (33 µM), suggesting tight coupling between ATP hydrolysis and 3’to 5’ translocation along the ssDNA. The ATP coupling stoichiometry suggests a 1-nt translocation step size; however, the translocation kinetic step size, the average distance translocated between consecutive rate-limiting translocation steps, is larger (~4 nts at saturating [ATP]) and it changes with both ATP and salt concentration. The larger kinetic step size and the small ATP coupling stoichiometry could reflect a discontinuous stepping mechanism where the UvrD monomer translocates in rapid, 1-nt steps each hydrolyzing one ATP followed by a slower step that limits the overall rate of translocation; however, the ATP dependent change in the kinetic step size is not entirely consistent with this model. Rather the ATP and salt dependence changes in the translocation kinetic step size, suggest the presence of kinetically different populations of UvrD on the ssDNA. Translocation experiments on partial DNA duplex substrates with a 5’ flanking ssDNA tail also indicate the presence of kinetically different populations of UvrD, where UvrD that specifically initiates translocation from the ss/ds junction has a significantly smaller kinetic step size than UvrD initiating translocation from internal sites of the ssDNA tail. Interestingly, UvrD monomer specificity for binding to a 5’-ss/dsDNA junction, which orients UvrD to translocate away from the junction, suggest such iiijunctions may serve to specifically load the UvrD monomer on DNA where the ssDNA translocase activity of the monomer is required. This could be important for UvrD mediated RecA displacement from ssDNA at damaged DNA replication forks. Another interesting observation is that the UvrD monomer translocation rate is dependent on the ssDNA base composition where translocation was faster on pyrimidines than purines in the following order: dT\u3edC\u3edA\u3edG. The translocation rate decreased only 2-fold when changing from a ssDNA composed entirely of dT to a ssDNA composed of an equal mixture of each base. The ssDNA base specific effect on the translocation rate suggests the ssDNA base is an important element of the ssDNA recognized by UvrD during translocation, as suggested by the UvrD crystal structure. This is further supported by studies using abasic sites and polyethylene glycol spacers in the ssDNA where the absence of the ssDNA base has a greater effect on UvrD processivity than the absence of the ssDNA deoxyribose and phosphate moieties. Based on these results an inchworm stepping model for UvrD monomer translocation along ssDNA is proposed that is consistent with crystallographic studies of UvrD and PcrA; however, the presence of kinetic heterogeneity in the UvrD population adds an additional level of complexity to the mechanism, which cannot be assessed from static structures