Visualization and measurement of DNA methyltransferase activity in living cells.
In this unit, a live-cell assay to measure DNA (cytosine-5) methyltransferase (MTase) activity at the single-cell level is described. This method takes advantage of the irreversible binding of enzymatically active MTases to genomic DNA substituted with the mechanism-based inhibitor 5-aza-2'-deo...
| Autors principals: | , , |
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| Format: | Journal article |
| Idioma: | English |
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2008
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| _version_ | 1826299787631132672 |
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| author | Schermelleh, L Spada, F Leonhardt, H |
| author_facet | Schermelleh, L Spada, F Leonhardt, H |
| author_sort | Schermelleh, L |
| collection | OXFORD |
| description | In this unit, a live-cell assay to measure DNA (cytosine-5) methyltransferase (MTase) activity at the single-cell level is described. This method takes advantage of the irreversible binding of enzymatically active MTases to genomic DNA substituted with the mechanism-based inhibitor 5-aza-2'-deoxycytidine (5-aza-dC). The procedure comprises incorporation of this nucleoside analog into DNA during replication and quantification of the time-dependent MTase immobilization by fluorescence recovery after photobleaching (FRAP). This trapping assay monitors kinetic properties and activity-dependent immobilization of MTases in their native environment and enables direct comparison of mutations and inhibitors that affect MTase regulation and catalytic activity in single living cells. In addition, a simplified protocol to obtain qualitative information on the activity of either endogenously or exogenously expressed MTases is provided. |
| first_indexed | 2024-03-07T05:07:16Z |
| format | Journal article |
| id | oxford-uuid:da595a03-724f-4ea7-a26d-22b541f08e20 |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-07T05:07:16Z |
| publishDate | 2008 |
| record_format | dspace |
| spelling | oxford-uuid:da595a03-724f-4ea7-a26d-22b541f08e202022-03-27T09:02:37ZVisualization and measurement of DNA methyltransferase activity in living cells.Journal articlehttp://purl.org/coar/resource_type/c_dcae04bcuuid:da595a03-724f-4ea7-a26d-22b541f08e20EnglishSymplectic Elements at Oxford2008Schermelleh, LSpada, FLeonhardt, HIn this unit, a live-cell assay to measure DNA (cytosine-5) methyltransferase (MTase) activity at the single-cell level is described. This method takes advantage of the irreversible binding of enzymatically active MTases to genomic DNA substituted with the mechanism-based inhibitor 5-aza-2'-deoxycytidine (5-aza-dC). The procedure comprises incorporation of this nucleoside analog into DNA during replication and quantification of the time-dependent MTase immobilization by fluorescence recovery after photobleaching (FRAP). This trapping assay monitors kinetic properties and activity-dependent immobilization of MTases in their native environment and enables direct comparison of mutations and inhibitors that affect MTase regulation and catalytic activity in single living cells. In addition, a simplified protocol to obtain qualitative information on the activity of either endogenously or exogenously expressed MTases is provided. |
| spellingShingle | Schermelleh, L Spada, F Leonhardt, H Visualization and measurement of DNA methyltransferase activity in living cells. |
| title | Visualization and measurement of DNA methyltransferase activity in living cells. |
| title_full | Visualization and measurement of DNA methyltransferase activity in living cells. |
| title_fullStr | Visualization and measurement of DNA methyltransferase activity in living cells. |
| title_full_unstemmed | Visualization and measurement of DNA methyltransferase activity in living cells. |
| title_short | Visualization and measurement of DNA methyltransferase activity in living cells. |
| title_sort | visualization and measurement of dna methyltransferase activity in living cells |
| work_keys_str_mv | AT schermellehl visualizationandmeasurementofdnamethyltransferaseactivityinlivingcells AT spadaf visualizationandmeasurementofdnamethyltransferaseactivityinlivingcells AT leonhardth visualizationandmeasurementofdnamethyltransferaseactivityinlivingcells |