A Helicase Assay Based on the Displacement of Fluorescent, Nucleic Acid-Binding Ligands

Eggleston, Angela K.; Rahim, Nazir; Kowalczykowski, Stephen C.

doi:10.1093/nar/24.7.1179

Cited by 82 publications

(80 citation statements)

References 48 publications

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“…6A) using the ensemble assay (Fig. S6) (16,42). At the lowest temperature tested (20°C), we did not observe any unwinding (Fig.…”

Section: Initiation Of Duplex Dna Unwinding Occurs Randomly On Dsdna Andmentioning

confidence: 81%

“…We used AF546 SSB, which possesses a fluorescent dye with spectral properties different from YO-PRO-1-stained dsDNA, and which afforded the potential advantage of observing DNA unwinding without needing to wash out the fluorescent DNAbinding dye. To ascertain whether the YO-PRO-1 interferes with RecQ helicase activity, we used an ensemble dye-displacement assay for DNA unwinding (42). RecQ unwinds DNA stained with YO-PRO-1 at 90% of the rate observed with Hoechst 33258, a DNA-binding dye that does not interfere with RecQ unwinding activity (16), and it unwinds DNA stained with ethidium bromide at ∼76% of that rate (Fig.…”

Section: Resultsmentioning

confidence: 99%

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Single-molecule visualization of RecQ helicase reveals DNA melting, nucleation, and assembly are required for processive DNA unwinding

Rad

Forget

Baskin

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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DNA helicases are motor proteins that unwind double-stranded DNA (dsDNA) to reveal single-stranded DNA (ssDNA) needed for many biological processes. The RecQ helicase is involved in repairing damage caused by DNA breaks and stalled replication forks via homologous recombination. Here, the helicase activity of RecQ was visualized on single molecules of DNA using a fluorescent sensor that directly detects ssDNA. By monitoring the formation and progression of individual unwinding forks, we observed that both the frequency of initiation and the rate of unwinding are highly dependent on RecQ concentration. We establish that unwinding forks can initiate internally by melting dsDNA and can proceed in both directions at up to 40-60 bp/s. The findings suggest that initiation requires a RecQ dimer, and that continued processive unwinding of several kilobases involves multiple monomers at the DNA unwinding fork. We propose a distinctive model wherein RecQ melts dsDNA internally to initiate unwinding and subsequently assembles at the fork into a distribution of multimeric species, each encompassing a broad distribution of rates, to unwind DNA. These studies define the species that promote resection of DNA, proofreading of homologous pairing, and migration of Holliday junctions, and they suggest that various functional forms of RecQ can be assembled that unwind at rates tailored to the diverse biological functions of RecQ helicase.recombination | fluorescence | TIRF microscopy | DNA repair | BLM

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“…6A) using the ensemble assay (Fig. S6) (16,42). At the lowest temperature tested (20°C), we did not observe any unwinding (Fig.…”

Section: Initiation Of Duplex Dna Unwinding Occurs Randomly On Dsdna Andmentioning

confidence: 81%

Section: Resultsmentioning

confidence: 99%

Single-molecule visualization of RecQ helicase reveals DNA melting, nucleation, and assembly are required for processive DNA unwinding

Rad

Forget

Baskin

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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“…Detection of heat shock protein 72 (HSP-72) immunoreactivity and cellular necrosis by acid fuchsin staining were carried out by previously described methods (19) in brains of 4 control rats and 4 rats 48 h after insulin-induced hypoglycemia. Additional brains from 4 control rats and 6 insulin-induced hypoglycemic rats were perfused 48 h later with paraformaldehyde, paraffin-embedded, sectioned, and stained with bis-benzimide (33258; Hoechst, Frankfurt, Germany), which binds to poly[d(A-T)] sequences in nuclear DNA (20). These sections were observed by fluorescent microscopy at an excitation/emission spectrum of 350/460 nm.…”

Section: Nc Tkacs and Associatesmentioning

confidence: 99%

Presumed apoptosis and reduced arcuate nucleus neuropeptide Y and pro-opiomelanocortin mRNA in non-coma hypoglycemia.

2000

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Hypoglycemia reduces sympathoadrenal responses to subsequent hypoglycemic bouts by an unknown mechanism. To assess whether such hypoglycemia-associated autonomic failure is due to actual brain damage, male Sprague-Dawley rats underwent 1-h bouts of insulininduced (5 U/kg i.v.) hypoglycemia (1.6-2.8 mmol/l) 1 or 3 times on alternate days. Rats remained alert and were rescued with intravenous glucose at 60-80 min. Plasma epinephrine and corticosterone responses were significantly reduced during the second and third bouts. Brains from these rats were processed by the terminal transferase-mediated deoxyuridine triphosphatebiotin nick end-labeling ( W ith the advent of intensive insulin therapy for type 1 diabetes, there has been an increasing incidence of hypoglycemic episodes (1). Some of these are severe, resulting in coma and subsequent permanent brain damage and/or death (2-5). Other less severe hypoglycemic episodes do not impair consciousness. Regardless of degree, hypoglycemia is associated with a robust counterregulatory response (CRR) (6-9). Low levels of plasma glucose are sensed by selective neurons in the brain (10-14) as well as by glucosensors in the hepatic portal vein (15). Signals from these sensors provoke activation of the sympathoadrenal system with preferential release of epinephrine. In addition, cortisol is released from the adrenal cortex and glucagon from the pancreatic ␣-cells (6-9,16). This response maximizes the mobilization of glucose in the body. In addition to this CRR, subjects develop an awareness of their hypoglycemic state (6,9). Both the CRR and the hypoglycemia awareness can become blunted after only a single bout of hypoglycemia (9,17). Hypoglycemia awareness recovers fairly rapidly but the diminished CRR is often prolonged (6), suggesting that different mechanisms are responsible for the two phenomena. However, the mechanism underlying either of these remains unclear.Here, we present a rat model of the dampened CRR and show that a single bout of moderate hypoglycemia produces highly site-specific apparent apoptotic response in cells of the hypothalamic arcuate nucleus (ARC). This apoptosis is associated with a significant reduction in the expression of both neuropeptide Y (NPY) and pro-opiomelanocortin (POMC) mRNA in this same nucleus. RESEARCH DESIGN AND METHODSAnimals and hypoglycemia. Male Sprague-Dawley rats were used at 300-400 g. They were housed at 22-23°C on a 12-h light-dark schedule and fed Purina rat chow (#5001) (PMI Nutrition International, Brentwood, MO) ad libitum. One group of rats (n = 12) had right atrial silastic catheters implanted via the right jugular vein under ketamine (45 mg/kg)/xylazine (9 mg/kg i.p.) anesthesia with buprenorphine (0.2 mg/kg) analgesia. Catheters were filled with a polyvinylpyrrolidone/heparin mix, which was replaced daily. The rats were allowed 4-5 days to recover and then were fasted overnight. Additional rats without venous catheters were also used (n = 34) and fasted overnight before testing. Between 0800 and 1000, a blood sample (...

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“…In contrast, the majority of DNA helicases require a short ssDNA overhang from which to initiate unwinding (13). The maximum rate of enzyme movement is measured between 1000 and 1500 bp s Ϫ1 at 37°C (4,14,15), which is much faster than related enzymes such as E. coli Rep and UvrD that translocate and unwind DNA at about 20 bp s Ϫ1 (16,17) in bulk phase measurements, but which can move as rapidly as 275 bp s Ϫ1 in single-molecule experiments (18).…”

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confidence: 97%

Bipolar DNA Translocation Contributes to Highly Processive DNA Unwinding by RecBCD Enzyme

Dillingham

Webb²,

Kowalczykowski

2005

Journal of Biological Chemistry

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We recently demonstrated that the RecBCD enzyme is a bipolar DNA helicase that employs two single-stranded DNA motors of opposite polarity to drive translocation and unwinding of duplex DNA. We hypothesized that this organization may explain the exceptionally high rate and processivity of DNA unwinding catalyzed by the RecBCD enzyme. Using a stopped-flow dye displacement assay for unwinding activity, we test this idea by analyzing mutant RecBCD enzymes in which either of the two helicase motors is inactivated by mutagenesis. Like the wild-type RecBCD enzyme, the two mutant proteins maintain the ability to bind tightly to blunt duplex DNA ends in the absence of ATP. However, the rate of forward translocation for the RecB motor-defective enzyme is only ϳ30% of the wild-type rate, whereas for the RecD motor-defective enzyme, it is ϳ50%. More significantly, the processivity of translocation is substantially reduced by ϳ25-and 6-fold for each mutant enzyme, respectively. Despite retaining the capacity to bind blunt dsDNA, the RecB-mutant enzyme has lost the ability to unwind DNA unless the substrate contains a short 5-terminated singlestranded DNA overhang. The consequences of this observation for the architecture of the single-stranded DNA motors in the initiation complex are discussed.The RecBCD enzyme of Escherichia coli is involved in the repair of double-stranded breaks in DNA (for review, see Ref. 1). This is an important function, because it has recently become apparent that double-stranded breaks occur frequently as a result, among other things, of replication through damaged template DNA (for discussions, see Refs. 2 and 3). The break in the nucleic acid is rescued by homologous recombination, which uses an undamaged copy of the DNA molecule as a template for the repair. Recombinational DNA repair involves several gene products, and the RecBCD helicase/nuclease acts at the initiation step. The enzyme binds tightly to blunt or nearly blunt DNA ends (4, 5). Then, in a reaction that requires ATP hydrolysis, RecBCD rapidly tracks along the duplex DNA, unwinding it as it goes, and predominantly degrading the 3Ј-terminated single strand into shorter fragments. This destructive, nucleolytic mode of action is modified when the enzyme encounters a correctly oriented DNA sequence called Chi (crossover hotspot instigator 5Ј-GCTGGTGG). This important regulatory sequence is over-represented in the E. coli genome and is particularly found clustered, with appropriate directionality, around the origin of replication (6, 7). Upon Chi recognition, degradation of the 3Ј-terminated strand is reduced, and degradation of the 5Ј-strand is up-regulated, although less so, while DNA translocation and unwinding continue (8 -10). Consequently, the product of the enzyme is a DNA duplex with a long 3Ј-terminated ssDNA 3 overhang onto which the enzyme loads RecA protein (11) to create a recombinogenic molecule, ready for the subsequent DNA strand exchange step of homologous recombination (3). In the absence of Chi recognition, RecBCD cont...

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A Helicase Assay Based on the Displacement of Fluorescent, Nucleic Acid-Binding Ligands

Cited by 82 publications

References 48 publications

Single-molecule visualization of RecQ helicase reveals DNA melting, nucleation, and assembly are required for processive DNA unwinding

Single-molecule visualization of RecQ helicase reveals DNA melting, nucleation, and assembly are required for processive DNA unwinding

Presumed apoptosis and reduced arcuate nucleus neuropeptide Y and pro-opiomelanocortin mRNA in non-coma hypoglycemia.

Bipolar DNA Translocation Contributes to Highly Processive DNA Unwinding by RecBCD Enzyme

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