We thank the cross-council New Dynamics of Ageing Programme for funding this work [ES/G037086/1] and Prof Dave Sherratt (Department of Biochemistry, University of Oxford) for access to the Fuji FLA-3000.

1. Preparation of Substrate Oligonucleotides
<ol>
	<li>Synthesize custom oligonucleotides.
	<ol>
		<li>Design one single-stranded oligonucleotide with a 5&#39;-conjugated fluorescein molecule; this will be the backbone for every substrate. (Note that this is for a 3&#39;-5&#39; exonuclease; for a 5&#39;-3&#39; polarity exonuclease use a 3&#39;-conjugated backbone strand). If polarity is not known, both 3&#39; and 5&#39; should be tested.</li>
		<li>Design complementary strands such that when annealed to the fluorescently labeled oligonucleotide, the duplex substrate(s) of choice are formed. Note that the complementary strand is not conjugated to a fluorophore. When ordering, specify PAGE-purification (desalting is not sufficient).</li>
		<li>Store oligonucleotide stocks buffered in 10 mM at 1 nmol/&#956;l at &#8722;20 &#176;C and in the dark.</li>
	</ol>
	</li>
	<li>Mix the backbone labeled oligonucleotide with each complementary unlabeled oligonucleotide in a 1:1.2 molar ratio of labeled:unlabeled in buffer (10 mM Tris-HCl pH 7.6, 1 mM EDTA pH 8.0, 50 mM NaCl). (Note: a 1:1 ratio is most desirable but it is more important that ALL of the labeled strand is bound. Ratios of 1:1 - 1:1.5 can work.)</li>
	<li>Heat a waterbath or heatblock to 95 &#176;C and place the annealing mixture into this for 3 min then switch off and allow the whole to cool slowly (1 &#176;C/min) to below 30 &#176;C.</li>
	<li>Check full annealing of the labeled oligonucleotide by running the resulting duplex substrate on a native 10% polyacrylamide gel (10% 19:1 acrylamide:bis-acrylamide in 1x Tris-Borate-EDTA (TBE, 89 mM Tris base, 89 mM boric acid, 2 mM EDTA) polymerized with 0.05% w/v ammonium persulfate (APS) and 0.005% v/v TEMED).Visualize via PhosphorImager (see section 5 below).</li>
</ol>
2. Preparation of Acrylamide Gels
<ol>
	<li>Set up the gel casting system e.g. Hoefer SE400 gel rig with 16 cm x 18 cm plates and 1.5 mm spacers.
	<ol>
		<li>Clean glass plates and spacers thoroughly using detergent and water. Wear clean gloves to avoid resoiling the plates with oil from fingers, which would hamper consistent gel formation.</li>
		<li>Rinse the plates with plenty of distilled (deionized) water to remove detergent residue, then wipe with lint-free tissue (e.g. Kimwipes).</li>
		<li>Finally, spray with 70% ethanol and wipe - glass plates should squeak if they are clean. Set aside upright to allow the last of the ethanol to evaporate.</li>
		<li>Clean the rest of the gel forming equipment with distilled water (use detergent first if necessary) and dry.</li>
		<li>Assemble the glass plates and spacers into the gel-forming rig, making sure the system is leakproof.</li>
	</ol>
	</li>
	<li>Prepare the gel solution to the desired polyacrylamide percentage
	<ol>
		<li>For nuclease assay, use denaturing 14% polyacrylamide gels with 8 M urea in 1x TBE.</li>
		<li>To make 100 ml (enough for 2 gels with plenty of excess for leakage) take 48.05 g urea in a wide beaker and add 35 ml 40% 19:1 acrylamide solution and 10 ml 10x TBE (890 mM Tris, 890 mM boric acid, 20 mM EDTA, pH 8.3 with NaOH). CAUTION: Wear gloves - acrylamide is a potent neurotoxin. Dissolve the urea using a stir bar and magnetic stir plate (this will take some time).</li>
		<li>When dissolved, add milliQ-deionized water to 100 ml. Filter-sterilize using a 0.22 &#956;m membrane. If not to be used immediately, degas briefly using suction equipment and store in the dark (will keep a few days). Do not refrigerate as the urea will precipitate.</li>
	</ol>
	</li>
	<li>Polymerize and cast the gel.
	<ol>
		<li>Take the urea-acrylamide and add 0.5 ml 10% (w/v) ammonium persulfate (APS, made up in distilled water) and 50 &#956;l TEMED. Swirl the container slowly to mix without imparting air (which will inhibit polymerization) and pour into the gel former. Act quickly or the gel may set before pouring is finished.</li>
		<li>Add the appropriate comb to form wells for sample loading (chosen according to the gel rig and sample number to be analyzed e.g. 15- or 29-tooth combs are suitable for use with the Hoefer gel rig). Check that no air bubbles are introduced under the comb teeth.</li>
		<li>Leave to set. Monitor for leakage. Full polymerization is retarded at the combs owing to contact with the atmosphere. When the wells are firm, the rest of the gel will be set.</li>
		<li>Remove the combs and rinse out unpolymerized acrylamide using water and a pipette.</li>
	</ol>
	</li>
	<li>Set up the gel for running.
	<ol>
		<li>Set the cast gel into the gel running system and add the appropriate amount of 1x TBE running buffer into the lower chamber and into the gel wells. The gel is now ready to be loaded.</li>
	</ol>
	</li>
</ol>
3. Preparation of His-tagged Recombinant DmWRNexo Protein
<ol>
	<li>Bacterial expression of DmWRNexo protein
	<ol>
		<li>Using an inoculation loop, collect bacteria by scraping over the surface of glycerol stocks maintained at -80 &#176;C (bacteria are E. coli T7 Iq LysY (NEB) transformed with pIVEX2.3d-DmWRNexo). Streak out onto LB-agar plates (10 g tryptone, 5 g yeast extract, 5 g NaCl, 15 g agar made up to 1 L with distilled water and pH adjusted to 7.4) with 50 &#956;g/ml ampicillin. Incubate inverted overnight at 37 &#176;C.</li>
		<li>Pick a single colony from the plate into 50 ml LB (10 g tryptone, 5 g yeast extract, 5 g NaCl made up to 1 L with distilled water) supplemented with 50 &#956;g/ml ampicillin (made up in water and filter-sterilized to 0.22 &#956;m). Grow with shaking at 220 rpm 37 &#176;C overnight.</li>
		<li>Inoculate 1.8 L (aliquots of 600 ml each in three 2 L&#160;conical flasks to ensure adequate aeration) of LB-ampicillin with the overnight 50 ml culture and grow to OD600 ~ 0.8. Add IPTG to a final concentration of 0.7 mM and incubate at 26 &#176;C (to prevent protein aggregation) with shaking for 4-8 hr.</li>
		<li>Harvest the cell pellet by centrifugation at 8,000 rpm 4 &#176;C for 10 min - use 6 x 500 ml sterile plastic centrifuge bottles with 300 ml cell suspension in each. Discard supernatant and resuspend the pellet in a small volume (10 ml) of the original LB-amp , transfer to a 15 ml Falcon tube and then respin at 5,000 rpm to pellet the cells - remove the supernatant again and freeze at -80 &#176;C&#160;(this helps with lysis).</li>
	</ol>
	</li>
	<li>Bacterial cell lysis
	<ol>
		<li>Make up 3.2 ml lysis buffer containing 1 ml 5 M NaCl, 1 ml 1 M DTT, 200 &#956;l 1 M benzamidine and 1 ml 10 mg/ml lysozyme (resuspended in distilled water).</li>
		<li>To each pellet derived from 300 ml bacterial suspension, add 320 &#956;l lysis buffer. Resuspend as much as possible - use a &#39;sawn-off&#39; p1000 tip and stir as well as pipetting up and down. Incubate on ice 20 min. Add a further 100 &#956;l lysis buffer and mix well then transfer as much as possible of the resuspended lysate into a microfuge tube (it will be very viscous and hard to pipette).</li>
		<li>Centrifuge the lysate at 14,000 rpm in a precooled microfuge rotor at 4 &#176;C for 30 min, then take the supernatant to a clean microfuge tube: this contains the recombinant protein.</li>
	</ol>
	</li>
	<li>His-trap purification
	<ol>
		<li>Preparation of reagents: Label 5 microfuge tubes for each of the following concentrations of imidazole: 40 mM, 60 mM, 100 mM, 300 mM, and 500 mM (e.g. label 40-1, 40-2, 40-3, etc.), which will be used to collect the column fractions. Store the tubes on ice in a rack with lids open (but cover loosely with clingfilm or similar to keep clean whilst preparing everything else). Make up 25 ml of 20 mM imidazole and 10 ml stocks of each of the other concentrations of imidazole in 25 ml universal tubes, using 1x PBS (8 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4, 0.24 g KH2PO4, pH adjusted to 7.4 and made up to 1 L with distilled water) as the diluent.</li>
		<li>Adjust the bacterial lysate to 20 mM imidazole using an appropriate volume of 2&#160;M imidazole stock solution, and adjust volume to at least 1 ml (&#60;5 ml) using 20 mM imidazole.</li>
		<li>Wash a 1 ml His-Trap column (Amersham) with 10 ml MilliQ water by using a 5 or 10 ml syringe with Luer lock and gentle manual pressure, then equilibrate the column by passing through 10 ml of 20 mM imidazole made up in PBS (see step&#160;3.3.1 above).</li>
		<li>Load the lysate onto the column. As the lysate is loaded onto the column, significant back-pressure may be experienced; this is perfectly normal. Do not be tempted to apply too much force to the syringe, and maintain a steady even pressure. It is usual to see a mild color change as the lysate enters the column. Collect the flow through in a Sterilin bijou bottle (7 ml capacity) and store on ice.</li>
		<li>Wash the column with 10 ml of 20 mM imidazole and collect this in a 25 ml universal tube on ice, labeled as &#39;wash&#39;.</li>
		<li>Pass 5 ml of 40 mM imidazole through the column, collecting 1 ml fractions into each of the microfuge tubes (labeled 40.1, 40.2, 40.3, etc.): watch the plunger of the syringe carefully as this allows measurement of 1 ml.</li>
		<li>Now sequentially pass through 5 ml of each of 60 mM, 100 mM, 300 mM, and 500 mM imidazole, again collecting 5 x 1 ml fractions at each salt concentration.</li>
	</ol>
	</li>
	<li>Analysis of column fractions
	<ol>
		<li>Dot blot: Dot 2 &#956;l of each column fraction (and also load, flow through and wash) onto a sheet of nitrocellulose&#160;- it is easier if this is preruled with a grid using a soft pencil). Allow to air dry. Process the dot blot by blocking for 15 min to 1 hr room temperature in 5% nonfat dried milk made up in PBS with 0.2% v/v Tween 20, then wash in PBS-Tween several times and incubate with HRP-anti-his antibody&#160;diluted 1:500 in PBS-Tween for 30-60 min. Wash extensively with PBS-Tween then PBS, and detect bound antibody using enhanced chemiluminescence (ECL), visualized on Hyperfilm MP. This dot blot gives an excellent idea of peak fractions prior to SDS-PAGE analysis, and can inform subsequent purification steps if necessary (e.g. if protein stability is an issue, it is possible to pool peak fractions based on dot blot results and load straight onto gel filtration columns - see step 3.5 below).</li>
		<li>SDS-PAGE: Collect 50 &#956;l of each column fraction into a labeled microfuge tube containing 50 &#956;l 2x SDS-PAGE loading dyes (4% w/v SDS, 20% v/v glycerol, 120 mM Tris-HCl pH 6.8 with 200 mM DTT). Boil these samples for 5 min at 95-100 &#176;C (note if using a hot block without a heated lid, make a small hole in the lid of each tube with a narrow gauge syringe needle to prevent the tubes &#39;exploding&#39; as they heat up). Run 10% SDS-PAGE minigels loading 10 &#956;l of each column fraction and stain with Coomassie brilliant blue to verify peak fractions and % purity of the protein. If required, it is also possible to western blot for his-tagged protein by transferring proteins separated by SDS-PAGE onto nitrocellulose; block the membranes with 5% nonfat milk in PBS-0.2% Tween 20, then wash and probe with HRP-conjugated anti-his antibody (1:500) as for the dot blot.</li>
	</ol>
	</li>
	<li>To increase purity, the peak fractions from the his-trap column can be pooled and loaded onto a Superdex 75 gel filtration column with elution in increasing salt (as previously described Boubriak&#160;et al.13 and&#160;Mason et al.14).</li>
</ol>
4. Nuclease Assay
<ol>
	<li>Make working stocks of substrates.
	<ol>
		<li>Use 20 pmol substrate per reaction (though this can be varied according to the question to be addressed) so a convenient working stock is 200 pmol/&#956;l (i.e. 10x). Working stocks are made up in small volumes of annealing buffer (10 mM Tris-HCl pH7.6, 1 mM EDTA pH 8.0, 50 mM NaCl) or in 10 mM Tris-HCl pH 7.0 (up to pH 8.5, according to nuclease) without salt or EDTA, according to enzyme sensitivity or assay to be performed .</li>
		<li>Keep at 4 &#176;C and in the dark (wrapped in foil). Substrates last for many weeks with no loss of duplex integrity or of fluorescent signal.</li>
	</ol>
	</li>
	<li>Assay exonuclease protein concentration by colorimetric analysis, or UV spectrometry at Abs260.
	<ol>
		<li>Store in small aliquots at -80 &#176;C.</li>
		<li>If samples are frozen/thawed once, assume a protein misfold rate of 5% (or define empirically for each protein to be assayed) and adjust concentrations accordingly.</li>
		<li>Discard proteins for assay of exonuclease activity after two freeze/thaw cycles, and note status of protein for each experiment.</li>
	</ol>
	</li>
	<li>Design experiment and work out master mix (MM) quantities for samples.
	<ol>
		<li>Make a MM containing all the components common to the samples being tested (usually everything except the enzyme or substrate) to decrease inter-sample variation.</li>
		<li>Allow for pipetting error when making up the MM e.g. calculate volumes for N samples by making sufficient for (N + 0.1N) samples.</li>
		<li>Set up reactions in a total volume of 20 &#956;l/sample, containing 1 pmol of enzyme and 20 pmol of substrate (as default before optimization - obviously amounts vary according to what is being tested e.g. alter amount of enzyme if testing effect of enzyme concentration). For DmWRNexo, the optimal buffer for nuclease activity was found empirically to contain 40 mM Tris-HCl pH 8.0, 4 mM MgCl2, 5 mM dithiothreitol, with 0.1 mg/ml molecular biology grade BSA.</li>
	</ol>
	</li>
	<li>Set up reactions.
	<ol>
		<li>Keep all reagents at 4 &#176;C (ice bucket or cryorack).</li>
		<li>Make up master mix and aliquot into the appropriate number of sterile DNase-free 0.5 ml microfuge tubes. Include a no-enzyme control to visualize the initial substrate (nondegraded control).</li>
		<li>Set a heat block to the appropriate temperature for enzyme activity (37 &#176;C, with a final time point of 30 min was optimal for DmWRNexo though this may need to be determined empirically by comparing activity of the chosen enzyme at a range of temperatures and times). Potential enzyme inhibitors commonly present in buffers should be carefully tested for (e.g. EDTA inhibits DmWRNexo, as does excess ATP14, presumably by chelation of the Mg2+ critical for nuclease activity). Note that even high concentrations of imidazole (&#62;300 mM) have no impact on either DmWRNexo enzyme activity nor does it quench fluorescence.</li>
		<li>Make up STOP solution (80% Formamide, 1x TBE). This is at 3x. This may contain dyes such as bromophenol blue if required. Store at RT.</li>
		<li>Set up the following near the heat block: the final reagent(s) to be added to the reaction mix (usually enzyme); a 1-10 &#956;l pipettor and tips, a timer, cryorack or ice bucket, and STOP solution.</li>
	</ol>
	</li>
	<li>Start the reaction.
	<ol>
		<li>Set the timer going on addition of enzyme to the first tube. Place the tube in the heat block.</li>
		<li>Continue adding enzyme to the rest of the tubes at appropriate intervals. (15 sec is ample for simple reactions that only require STOP solution to complete). Place in heat block as soon as enzyme has been added.</li>
	</ol>
	</li>
	<li>Stop the reaction.
	<ol>
		<li>As the timer reaches the correct time (for example 15 min) add 10 &#956;l STOP solution to the first sample and mix by pipetting. Place immediately on ice/in the cryorack.</li>
		<li>Continue to stop the reactions at the appropriate time intervals and in the same order as they were set up so that each reaction has had exactly the same incubation time.</li>
	</ol>
	</li>
	<li>The samples are now ready to load on the prepared denaturing gel.
	<ol>
		<li>Load half (or all) of each sample using gel-loading tips for accurate pipetting. At the levels of oligonucleotide used in the reaction, half the reaction will give good output for visualization and allows for a repeat to be run (e.g. if the initial gel does not run correctly).</li>
		<li>It is convenient to load 1x STOP solution containing dye (e.g. bromophenol blue) in one or more lanes to ascertain how the gel is running.</li>
		<li>Fluorescent oligonucleotides of known sizes may be run alongside the samples as a size marker.</li>
	</ol>
	</li>
	<li>Run the gel.
	<ol>
		<li>Add the top running buffer chamber to the gel apparatus and fill with running buffer (1x TBE), making sure the samples are not disturbed. Check for buffer leakage.</li>
		<li>Connect the gel rig to a power supply and run at 200 V (constant voltage) for 150-200 min or until the bromophenol blue dye (if using) is around two-thirds of the way down the gel (it runs around the level of 10nt DNA in this percentage gel). Monitor for buffer leakage, which can skew or prevent separation.</li>
		<li>Stop the gel and remove from the running apparatus.</li>
	</ol>
	</li>
</ol>
5. Gel Analysis and Imaging
<ol>
	<li>Determine the sensitivity of the Imager to be used, e.g. the Fuji FLA-3000 PhosphorImager/Fluorescence Imager can detect fluorescence of fluorescein using a blue 473 nm laser, using its greatest sensitivity setting of F1000 and the smallest pixel read (resolution) of 50 &#956;m.</li>
	<li>Scan the gel.
	<ol>
		<li>Remove the spacers and one gel plate carefully. Rinse the gel remaining on the other gel plate thoroughly (but carefully) to remove traces of precipitated urea that will otherwise interfere with scanning.</li>
		<li>Set the plate (with the gel still attached) gel-down onto the glass face of the gel-holder and place it into the Imager. Note the coordinates of the gel on the glass (if it does not fill the whole surface).</li>
		<li>Open the appropriate software on the connected computer and scan the noted region of the scanning area where the gel is. Use the best conditions determined as in step 5.1. The resulting file will be relatively large (several MB) but the image can be cropped and compressed if needed.</li>
		<li>Save, then export as a file that can be read by analysis software e.g. export as a .tiff for analysis using ImageJ.</li>
	</ol>
	</li>
	<li>Analyze the data.
	<ol>
		<li>A successful exonuclease assay will cause degradation of the substrate sequentially along the oligonucleotide towards the fluorophore, resulting in a characteristic ladder-like pattern of fluorescent DNA fragments on the gel. Species with higher mobility on the gel (which run further) are smaller than the nondegraded starting substrate and therefore represent products of exonuclease degradation.</li>
		<li>Use the &#39;Gels&#39; tool in ImageJ to quantify the relative intensities of species in each lane dependent upon their mobility, in order to determine the levels of degradation hence exonuclease activity in each sample.</li>
	</ol>
	</li>
</ol>

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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Drosophila+orthologue+of+progeroid+human+WRN+exonuclease,+DmWRNexo,+cleaves+replication+substrates+but+is+inhibited+by+uracil+or+abasic+sites+:+Analysis+of+DmWRNexo+activity+in+vitro.">The Drosophila orthologue of progeroid human WRN exonuclease, DmWRNexo, cleaves replication substrates but is inhibited by uracil or abasic sites : Analysis of DmWRNexo activity in vitro.</a> Age (Dordr). , (2012).</li><li>Machwe, A., Xiao, L., Orren, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Length-dependent+degradation+of+single-stranded+3'+ends+by+the+Werner+syndrome+protein+(WRN):+implications+for+spatial+orientation+and+coordinated+3'+to+5'+movement+of+its+ATPase/helicase+and+exonuclease+domains.">Length-dependent degradation of single-stranded 3' ends by the Werner syndrome protein (WRN): implications for spatial orientation and coordinated 3' to 5' movement of its ATPase/helicase and exonuclease domains.</a> BMC Mol. Biol. 7, 6 (2006).</li><li>Mangerich, A., Veith, S., Popp, O., Fahrer, J., Martello, R., Bohr, V. A., Burkle, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+WRN+exonuclease+activity+by+isotope+dilution+mass+spectrometry.">Quantitative analysis of WRN exonuclease activity by isotope dilution mass spectrometry.</a> Mech. Ageing Dev. 133, 575-579 (2012).</li><li>Xue, Y., Ratcliff, G. C., Wang, H., Davis-Searles, P. R., Gray, M. D., Erie, D. A., Redinbo, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+minimal+exonuclease+domain+of+WRN+forms+a+hexamer+on+DNA+and+possesses+both+3'-+5'+exonuclease+and+5'-protruding+strand+endonuclease+activities.">A minimal exonuclease domain of WRN forms a hexamer on DNA and possesses both 3'- 5' exonuclease and 5'-protruding strand endonuclease activities.</a> Biochemistry. 41, 2901-2912 (2002).</li><li>Machwe, A., Ganunis, R., Bohr, V. A., Orren, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+blockage+of+the+3'--&gt;5'+exonuclease+activity+of+WRN+protein+by+certain+oxidative+modifications+and+bulky+lesions+in+DNA.">Selective blockage of the 3'--&gt;5' exonuclease activity of WRN protein by certain oxidative modifications and bulky lesions in DNA.</a> Nucleic Acids Res. 28, 2762-2770 (2000).</li></ol>

The authors have nothing to disclose.

Determination of exonuclease activity of purified proteins requires the analysis of DNA cleavage products. Sequential cleavage of DNA by exonucleases can be visualized by separation of labeled cleavage products on acrylamide gels. Historically this involved end-labeling of the DNA substrate with a radiolabel (e.g. 32P or 35S), but with the disadvantages inherent in use of radiolabel (cost, safety issues, and instability over time). To overcome these problems, we have developed an exonucleas...

Nucleases serve a vital role in cells in removing damaged DNA, resolving nonduplex structures such as Holliday junctions and providing proof-reading capacity during DNA replication, both intrinsic within DNA polymerases and extrinsic to them1. Nucleases can act either by sequentially degrading DNA from free ends (exonucleases) or by cleaving internal phosphodiester bonds within a longer DNA molecule (endonucleases). Loss of nuclease activity can result in highly specific genome instability phenotypes. While mutation of the RecQ helicase family member BLM result in excessively high rates of sister chromatid exchange and globally elevated cancer rates (reviewed byPayne and Hickson2), mutation of the highly related WRN protein leads to premature aging3; the major significant difference between these two family members is the presence of a 3&#39;-5&#39; exonuclease domain with in WRN4. Evidence of a critical role of the WRN exonuclease in maintaining genome stability has accumulated from analysis of genotypes in WS patients5, together with point mutation and deletion studies in human cells, backed by crystallographic studies of the isolated exonuclease domain6. However, cooperation and cross talk between WRN&#39;s exonuclease activity and its central helicase activity7 makes it difficult to tease apart the functionality of each and their relative contributions to genome stability. In plants and lower metazoan animals, WRN exonuclease activity is present on a single polypeptide lacking helicase activity8-10 (reviewed in&#160;Cox and Boubriak11); it has been demonstrated biochemically in Arabidopsis that this exonuclease acts coordinately with the cognate WRN helicase, effectively reconstituting the combined enzyme activities observed in vertebrate WRN9. We have studied WRN exonuclease in Drosophila since the excellent genetic tools allow analysis of the impact of exonuclease mutation (without impacting on the presumptive cognate helicase) at the whole organism level and through development10,12. Moreover, we have cloned, expressed, and purified recombinant Drosophila WRN exonuclease (DmWRNexo) allowing full biochemical analysis of its enzyme properties13,14.
Nuclease analysis in vitro has traditionally been conducted using radiolabeled oligonucleotides, assessing degradation by looking for laddering of products on acrylamide gels4,8,15. While sensitive, such assays are not quantitatively reproducible day-to-day because of radioactive decay of the labeled substrates. Additionally, handling and disposal of radioactive reagents pose significant environmental and health issues; sourcing of radiolabel is also becoming increasingly problematic. An alternative recent method assesses the amount of the final degradation product by mass spectrometry16. However, it is time consuming (taking several days), requires specialized equipment, and the readout is the amount of end product (single nucleotide) so is not suitable for sensitive measurement of aspects such as enzyme processivity or for determining whether some nucleotides, sequences, or modifications lead to nuclease pausing or halt. To overcome these problems, we have adapted the traditional gel-based assays for use with fluorescent oligonucleotide substrates, generating stably labeled substrates that can be used reproducibly over long time periods and thus allow direct comparison of nuclease activities under different conditions.

<table><tbody><tr><td colspan="4">&lt;strong&gt;Reagent/Material&lt;/strong&gt;</td></tr><tr><td>Custom oligonucleotides</td><td>Eurogentec</td><td></td><td>It is necessary to obtain these at high purity &lt;em&gt;e.g.&lt;/em&gt; with PAGE purification.</td></tr><tr><td></td><td></td><td>5&#039;FLO</td><td>fluoresescein-5&#039; GAACTATGGCTCTC&lt;br/&gt; GAGTGCTAGGACATGTCTGA&lt;br/&gt; CTACGTACAAGTCACC - 3&#039;</td></tr><tr><td></td><td></td><td>bubble</td><td>5&#039;- GGTGACTTGTACGT&lt;br/&gt; AGTCAGACATGTCCTAGCAC&lt;br/&gt; TCGAGAGCCATAGTTC-3&#039;</td></tr><tr><td>40% 19:1 Acrylamide solution</td><td>Severn Biotech</td><td>20-2400-05</td><td>CAUTION: potent neurotoxin so gloves should be worn at all times</td></tr><tr><td>His-Trap columns (1 ml)</td><td>GE Healthcare</td><td>17-5247-01</td><td></td></tr><tr><td>All other reagents</td><td>any reputable supplier</td><td></td><td>Molecular biology grade is necessary (DNase-free); microfuge tubes similarly should be DNase- and RNase-free</td></tr><tr><td colspan="4">&lt;strong&gt;Equipment&lt;/strong&gt;</td></tr><tr><td>Hoefer SE400 gel apparatus</td><td>Hoefer</td><td>SE400-15-1.5</td><td></td></tr><tr><td>FLA-3000 (phosphor and fluorescence imager)</td><td>Fuji</td><td></td><td></td></tr><tr><td>Image Reader V2.02</td><td>FujiFilm</td><td></td><td></td></tr><tr><td>Image Gauge V3.3</td><td>FujiFilm</td><td></td><td></td></tr></tbody></table>

a fluorescence-based exonuclease assay to characterize dmwrnexo, orthologue of human progeroid wrn exonuclease, and its application to other nucleases

WRN exonuclease is involved in resolving DNA damage that occurs either during DNA replication or following exposure to endogenous or exogenous genotoxins. It is likely to play a role in preventing accumulation of recombinogenic intermediates that would otherwise accumulate at transiently stalled replication forks, consistent with a hyper-recombinant phenotype of cells lacking WRN. In humans, the exonuclease domain comprises an N-terminal portion of a much larger protein that also possesses helicase activity, together with additional sites important for DNA and protein interaction. By contrast, in Drosophila, the exonuclease activity of WRN (DmWRNexo) is encoded by a distinct genetic locus from the presumptive helicase, allowing biochemical (and genetic) dissection of the role of the exonuclease activity in genome stability mechanisms. Here, we demonstrate a fluorescent method to determine WRN exonuclease activity using purified recombinant DmWRNexo and end-labeled fluorescent oligonucleotides. This system allows greater reproducibility than radioactive assays as the substrate oligonucleotides remain stable for months, and provides a safer and relatively rapid method for detailed analysis of nuclease activity, permitting determination of nuclease polarity, processivity, and substrate preferences.

Performing in vitro analysis of exonuclease activity requires a number of preparatory steps in addition to the actual analysis. An overview of the procedures is shown in Figure 1. 
Prior to conducting fluorescence-based exonuclease assays, it is critical to optimize detection of the fluorescently labeled oligonucleotide substrate following separation on urea-acrylamide gels using a suitable fluorescence imaging system. Filter choice is extremely important as this can ...

Watch this Scientific Journal Video about A Fluorescence-based Exonuclease Assay to Characterize DmWRNexo, Orthologue of Human Progeroid WRN Exonuclease, and Its Application to Other Nucleases at JoVE

A Fluorescence-based Exonuclease Assay to Characterize DmWRNexo, Orthologue of Human Progeroid WRN Exonuclease, and Its Application to Other Nucleases

Exonucleases play critical roles in ensuring genome stability. Loss of WRN exonuclease function results in premature aging. Studying substrates and other requirements of the nuclease in vitro can help elucidate its role in vivo. Here we demonstrate a rapid and reproducible fluorescence-based assay to measure its nuclease activity.

WRN exonuclease is involved in resolving DNA damage that occurs either during DNA replication or following exposure to endogenous or exogenous genotoxins. ...

a-fluorescence-based-exonuclease-assay-to-characterize-dmwrnexo

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

5.0K 次观看.  University of Oxford. Exonucleases play critical roles in ensuring genome stability. Loss of WRN exonuclease function results in premature aging. Studying substrates and other requirements of the nuclease in vitro can help elucidate its role in vivo. Here we demonstrate a rapid and reproducible fluorescence-based assay to measure its nuclease activity.

视频: A Fluorescence-based Exonuclease Assay to Characterize DmWRNexo, Orthologue of Human Progeroid WRN Exonuclease, and Its Application to Other Nucleases

Atomic Force Microscopy Investigations of DNA Lesion Recognition in Nucleotide Excision Repair

AFM imaging is a powerful technique for the study of protein-DNA interactions. This single molecule method allows the simultaneous resolution of different molecules and molecular assemblies in a heterogeneous sample. In the particular context of DNA interacting protein systems, different protein complex forms and their corresponding binding positions on target sites containing DNA fragments can thus be distinguished. Here, an application of AFM to the study of DNA lesion recognition in the prokaryotic and eukaryotic nucleotide excision DNA repair (NER) systems is presented. The procedures of DNA and protein sample preparations are described and experimental as well as analytical details of the experiments are provided. The data allow important conclusions on the strategies by which target site verification may be achieved by the NER proteins. Interestingly, they indicate different approaches of lesion recognition and identification for the eukaryotic NER system, depending on the type of lesion. Furthermore, distinct structural properties of the two different helicases involved in prokaryotic and eukaryotic NER result in and explain the different strategies observed for these two systems. Importantly, these experimental and analytical approaches can be applied not only to the study of DNA repair but also very similarly to other DNA interacting protein systems such as those involved in replication or transcription processes.

Here, the study of different DNA lesion recognition approaches via single molecule AFM imaging is demonstrated with the nucleotide excision repair system as an example. The procedures of DNA and protein sample preparations and experimental as well as analytical details for the AFM experiments are described.

原子力显微镜检查DNA损伤识别核苷酸切除修复

AFM imaging is a powerful technique for the study of protein-DNA interactions. This single molecule method allows the simultaneous resolution of different ...

科研

遗传学

DNA Polymerase Activity Assay Using Near-infrared Fluorescent Labeled DNA Visualized by Acrylamide Gel Electrophoresis

For any enzyme, robust, quantitative methods are required for characterization of both native and engineered enzymes. For DNA polymerases, DNA synthesis can be characterized using an in vitro DNA synthesis assay followed by polyacrylamide gel electrophoresis. The goal of this assay is to quantify synthesis of both natural DNA and modified DNA (M-DNA). These approaches are particularly useful for resolving oligonucleotides with single nucleotide resolution, enabling observation of individual steps during enzymatic oligonucleotide synthesis. These methods have been applied to the evaluation of an array of biochemical and biophysical properties such as the measurement of steady-state rate constants of individual steps of DNA synthesis, the error rate of DNA synthesis, and DNA binding affinity. By using modified components including, but not limited to, modified nucleoside triphosphates (NTP), M-DNA, and/or mutant DNA polymerases, the relative utility of substrate-DNA polymerase pairs can be effectively evaluated. Here, we detail the assay itself, including the changes that must be made to accommodate nontraditional primer DNA labeling strategies such as near-infrared fluorescently labeled DNA. Additionally, we have detailed crucial technical steps for acrylamide gel pouring and running, which can often be technically challenging.

This protocol describes the characterization of DNA polymerase synthesis of modified DNA through observation of changes to near-infrared fluorescently labeled DNA using gel electrophoresis and gel imaging. Acrylamide gels are used for high resolution imaging of the separation of short nucleic acids, which migrate at different rates depending on size.

用丙烯酰胺凝胶电泳法可视化近红外荧光标记 dna 聚合酶活性

For any enzyme, robust, quantitative methods are required for characterization of both native and engineered enzymes. For DNA polymerases, DNA synthesis ...

生物化学

Characterizing DNA Repair Processes at Transient and Long-lasting Double-strand DNA Breaks by Immunofluorescence Microscopy

The repair of double-stranded breaks (DSBs) in DNA is a highly coordinated process, necessitating the formation and resolution of multi-protein repair complexes. This process is regulated by a myriad of proteins that promote the association and disassociation of proteins to these lesions. Thanks in large part to the ability to perform functional screens of a vast library of proteins, there is a greater appreciation of the genes necessary for the double-strand DNA break repair. Often knockout or chemical inhibitor screens identify proteins involved in repair processes by using increased toxicity as a marker for a protein that is required for DSB repair. Although useful for identifying novel cellular proteins involved in maintaining genome fidelity, functional analysis requires the determination of whether the protein of interest promotes localization, formation, or resolution of repair complexes. 
The accumulation of repair proteins can be readily detected as distinct nuclear foci by immunofluorescence microscopy. Thus, association and disassociation of these proteins at sites of DNA damage can be accessed by observing these nuclear foci at representative intervals after the induction of double-strand DNA breaks. This approach can also identify mis-localized repair factor proteins, if repair defects do not simultaneously occur with incomplete delays in repair. In this scenario, long-lasting double-strand DNA breaks can be engineered by expressing a rare cutting endonuclease (e.g., I-SceI) in cells where the recognition site for the said enzyme has been integrated into the cellular genome. The resulting lesion is particularly hard to resolve as faithful repair will reintroduce the enzyme's recognition site, prompting another round of cleavage. As a result, differences in the kinetics of repair are eliminated. If repair complexes are not formed, localization has been impeded. This protocol describes the methodology necessary to identify changes in repair kinetics as well as repair protein localization.

Repair of double-strand DNA breaks is a dynamic process, requiring not only formation of repair complexes at the breaks, but also their resolution after the lesion is addressed. Here, we use immunofluorescence microscopy for transient and long-lasting double-stranded breaks as a tool to dissect this genome maintenance mechanism.

基于免疫荧光显微镜的瞬时和持久双链 dna 损伤 dna 修复过程的表征

The repair of double-stranded breaks (DSBs) in DNA is a highly coordinated process, necessitating the formation and resolution of multi-protein repair ...

癌症研究

Proofreading and DNA Repair Assay Using Single Nucleotide Extension and MALDI-TOF Mass Spectrometry Analysis

The maintenance of the genome and its faithful replication is paramount for conserving genetic information. To assess high fidelity replication, we have developed a simple non-labeled and non-radio-isotopic method using a matrix-assisted laser desorption ionization with time-of-flight (MALDI-TOF) mass spectrometry (MS) analysis for a proofreading study. Here, a DNA polymerase [e.g., the Klenow fragment (KF) of Escherichia coli DNA polymerase I (pol I) in this study] in the presence of all four dideoxyribonucleotide triphosphates is used to process a mismatched primer-template duplex. The mismatched primer is then proofread/extended and subjected to MALDI-TOF MS. The products are distinguished by the mass change of the primer down to single nucleotide variations. Importantly, a proofreading can also be determined for internal single mismatches, albeit at different efficiencies. Mismatches located at 2-4-nucleotides (nt) from the 3' end were efficiently proofread by pol I, and a mismatch at 5 nt from the primer terminus showed only a partial correction. No proofreading occurred for internal mismatches located at 6 - 9 nt from the primer 3' end. This method can also be applied to DNA repair assays (e.g., assessing a base-lesion repair of substrates for the endo V repair pathway). Primers containing 3' penultimate deoxyinosine (dI) lesions could be corrected by pol I. Indeed, penultimate T-I, G-I, and A-I substrates had their last 2 dI-containing nucleotides excised by pol I before adding a correct ddN 5'-monophosphate (ddNMP) while penultimate C-I mismatches were tolerated by pol I, allowing the primer to be extended without repair, demonstrating the sensitivity and resolution of the MS assay to measure DNA repair.

A non-labeled, non-radio-isotopic method for DNA polymerase proofreading and a DNA repair assay was developed by using high-resolution MALDI-TOF mass spectrometry and a single nucleotide extension strategy. The assay proved to be very specific, simple, rapid, and easy to perform for proofreading and repair patches shorter than 9-nucleotides.

单核苷酸延长和 MALDI 质谱分析校对及 DNA 修复方法

The maintenance of the genome and its faithful replication is paramount for conserving genetic information. To assess high fidelity replication, we have ...

Real-time Observation of the DNA Strand Exchange Reaction Mediated by Rad51

The DNA strand exchange reaction mediated by Rad51 is a critical step of homologous recombination. In this reaction, Rad51 forms a nucleoprotein filament on single-stranded DNA (ssDNA) and captures double-stranded DNA (dsDNA) non-specifically to interrogate it for a homologous sequence. After encountering homology, Rad51 catalyzes DNA strand exchange to mediate pairing of the ssDNA with the complementary strand of the dsDNA. This reaction is highly regulated by numerous accessary proteins in vivo. Although conventional biochemical assays have been successfully employed to examine the role of such accessory protein in vitro, kinetic analysis of intermediate formation and its progression into a final product has proven challenging due to the unstable and transient nature of the reaction intermediates. To observe these reaction steps directly in solution, fluorescence resonance energy transfer (FRET)-based real-time observation systems of this reaction were established. Kinetic analysis of real-time observations shows that the DNA strand exchange reaction mediated by Rad51 obeys a three-step reaction model involving the formation of a three-strand DNA intermediate, maturation of this intermediate, and the release of ssDNA from the mature intermediate. The Swi5-Sfr1 complex, an accessary protein conserved in eukaryotes, strongly enhances the second and third steps of this reaction. The FRET-based assays presented here enable us to uncover the molecular mechanisms through which recombination accessary proteins stimulate the DNA strand exchange activity of Rad51. The primary goal of this protocol is to enhance the repertoire of techniques available to researchers in the field of homologous recombination, particularly those working with proteins from species other than Schizosaccharomyces pombe, so that the evolutionary conservation of the findings presented herein can be determined.

Fluorescence resonance energy transfer-based real-time observation systems of the DNA strand exchange reaction mediated by Rad51 were developed. Using the protocols presented here, we are able to detect the formation of reaction intermediates and their conversion into products, while also analyzing the enzymatic kinetics of the reaction.

rad51 介导的 dna 链交换反应的实时观测

The DNA strand exchange reaction mediated by Rad51 is a critical step of homologous recombination. In this reaction, Rad51 forms a nucleoprotein filament ...

Single-Molecule Dwell-Time Analysis of Restriction Endonuclease-Mediated DNA Cleavage

This novel total internal reflection fluorescence microscopy-based assay facilitates the simultaneous measurement of the length of the catalytic cycle for hundreds of individual restriction endonuclease (REase) molecules in one experiment. This assay does not require protein labeling and can be carried out with a single imaging channel. In addition, the results of multiple individual experiments can be pooled to generate well-populated dwell-time distributions. Analysis of the resulting dwell-time distributions can help elucidate the DNA cleavage mechanism by revealing the presence of kinetic steps that cannot be directly observed. Example data collected using this assay with the well-studied REase, EcoRV - a dimeric Type IIP restriction endonuclease that cleaves the palindromic sequence GAT&#8595;ATC (where &#8595; is the cut site) - are in agreement with prior studies. These results suggest that there are at least three steps in the pathway to DNA cleavage that is initiated by introducing magnesium after EcoRV binds DNA in its absence, with an average rate of 0.17 s-1 for each step.

Using quantum-dot-labeled DNA and total internal reflection fluorescence microscopy, we can investigate the reaction mechanism of restriction endonucleases while using unlabeled protein. This single-molecule technique allows for massively multiplexed observation of individual protein-DNA interactions, and data can be pooled to generate well-populated dwell-time distributions.

限制性核酸内切酶介导的 DNA 切割的单分子停留时间分析

This novel total internal reflection fluorescence microscopy-based assay facilitates the simultaneous measurement of the length of the catalytic cycle for ...

Continuous Fluorescence-Based Endonuclease-Coupled DNA Methylation Assay to Screen for DNA Methyltransferase Inhibitors

DNA methylation, a form of epigenetic gene regulation, is important for normal cellular function. In cells, proteins called DNA methyltransferases (DNMTs) establish and maintain the DNA methylation pattern. Changes to the normal DNA methylation pattern are linked to cancer development and progression, making DNMTs potential cancer drug targets. Thus, identifying and characterizing novel small molecule inhibitors of these enzymes is of great importance. This paper presents a protocol that can be used to screen for DNA methyltransferase inhibitors. The continuous coupled kinetics assay allows for initial velocities of DNA methylation to be determined in the presence and absence of potential small molecule inhibitors. The assay uses the methyl-sensitive endonuclease Gla I to couple methylation of a hemimethylated DNA substrate to fluorescence generation.
This continuous assay allows for enzyme activity to be monitored in real time. Conducting the assay in small volumes in microtiter plates reduces the cost of reagents. Using this assay, a small example screen was conducted for inhibitors of DNMT1, the most abundant DNMT isozyme in humans. The highly substituted anthraquinone natural product, laccaic acid A, is a potent, DNA-competitive inhibitor of DNMT1. Here, we examine three potential small molecule inhibitors &#8212; anthraquinones or anthraquinone-like molecules with one to three substituents &#8212; at two concentrations to describe the assay protocol. Initial velocities are used to calculate the percent activity observed in the presence of each molecule. One of three compounds examined exhibits concentration-dependent inhibition of DNMT1 activity, indicating that it is a potential inhibitor of DNMT1.

DNA methyltransferases are potential cancer drug targets. Here, a protocol is presented to assess small molecules for DNA methyltransferase inhibition. This assay utilizes an endonuclease to couple DNA methylation to fluorescence generation and allows for enzyme activity to be monitored in real time.&#160;

基于荧光的连续核酸内切酶偶联DNA甲基化测定，用于筛选DNA甲基转移酶抑制剂

DNA methylation, a form of epigenetic gene regulation, is important for normal cellular function. In cells, proteins called DNA methyltransferases (DNMTs) ...

使用纳米荧光素酶报告基因检测定量 DNA 双链断裂修复

Assessment of DNA Double Strand Break Repair Activity Using High-throughput and Quantitative Luminescence-Based Reporter Assays

The repair of DNA double strand breaks (DSBs) is crucial for the maintenance of genome stability and cell viability. DSB repair (DSBR) in cells is mediated through several mechanisms: homologous recombination (HR), non-homologous end joining (NHEJ), microhomology-mediated end joining (MMEJ), and single strand annealing (SSA). Cellular assays are essential to measure the proficiency and modulation of these pathways in response to various stimuli.
Here, we present a suite of extrachromosomal reporter assays that each measure the reconstitution of a nanoluciferase reporter gene by one of the four major DSBR pathways in cells. Upon transient transfection into cells of interest, repair of pathway-specific reporter substrates can be measured in under 24 h by the detection of Nanoluciferase (NanoLuc) luminescence.
These robust assays are quantitative, sensitive, titratable, and amenable to a high-throughput screening format. These properties provide broad applications in DNA repair research and drug discovery, complementing the currently available toolkit of cellular DSBR assays.

作者聚焦：开发针对 DNA 损伤反应的新型抗癌疗法

We present protocols for the assessment of double strand break repair pathway proficiency in cells using a suite of luminescence-based extrachromosomal reporter substrates.

A Fluorescence-based Exonuclease Assay to Characterize DmWRNexo, Orthologue of Human Progeroid WRN Exonuclease, and Its Application to Other Nucleases

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原子力显微镜检查DNA损伤识别核苷酸切除修复

用丙烯酰胺凝胶电泳法可视化近红外荧光标记 dna 聚合酶活性

基于免疫荧光显微镜的瞬时和持久双链 dna 损伤 dna 修复过程的表征

单核苷酸延长和 MALDI 质谱分析校对及 DNA 修复方法

rad51 介导的 dna 链交换反应的实时观测

限制性核酸内切酶介导的 DNA 切割的单分子停留时间分析

基于荧光的连续核酸内切酶偶联DNA甲基化测定，用于筛选DNA甲基转移酶抑制剂

使用基于高通量和定量发光的报告基因分析评估 DNA 双链断裂修复活性

使用基于高通量和定量发光的报告基因分析评估 DNA 双链断裂修复活性

使用基于高通量和定量发光的报告基因分析评估 DNA 双链断裂修复活性