A Fluorescence-based Exonuclease Assay to Characterize DmWRNexo, Orthologue of Human Progeroid WRN Exonuclease, and Its Application to Other Nucleases
Summary
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.
Abstract
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.
Introduction
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'-5' 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'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 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.
Protocol
Representative Results
Discussion
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…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| Reagent/Material | |||
| Custom oligonucleotides | Eurogentec | It is necessary to obtain these at high purity e.g. with PAGE purification. | |
| 5'FLO | fluoresescein-5' GAACTATGGCTCTC GAGTGCTAGGACATGTCTGA CTACGTACAAGTCACC – 3' |
||
| bubble | 5'- GGTGACTTGTACGT AGTCAGACATGTCCTAGCAC TCGAGAGCCATAGTTC-3' |
||
| 40% 19:1 Acrylamide solution | Severn Biotech | 20-2400-05 | CAUTION: potent neurotoxin so gloves should be worn at all times |
| His-Trap columns (1 ml) | GE Healthcare | 17-5247-01 | |
| All other reagents | any reputable supplier | Molecular biology grade is necessary (DNase-free); microfuge tubes similarly should be DNase- and RNase-free | |
| Equipment | |||
| Hoefer SE400 gel apparatus | Hoefer | SE400-15-1.5 | |
| FLA-3000 (phosphor and fluorescence imager) | Fuji | ||
| Image Reader V2.02 | FujiFilm | ||
| Image Gauge V3.3 | FujiFilm |
Referencias
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