Abstract
Single molecule sorting of the DNA repair machines (477.1)
The FASEB journal, Vol.28(S1), p.n/a
04/2014
DOI: 10.1096/fasebj.28.1_supplement.477.1
Abstract
The functional diversity and dynamic nature of the eukaryotic proteome are highly enhanced by posttranslational modifications (PTMs). DNA repair enzymes are often controlled by reversible PTMs that modulate their activities and interactions with their targets to broaden the range of repairable DNA lesions. A fraction of a DNA repair protein carrying an activating or modifying PTM may greatly influence cellular fate. To expedite the quantification, analysis and comparison of distinct posttranslationally modified pools of a protein we developed a technique to simultaneously, but separately evaluate the activities of modified and unmodified proteins extracted from the human cells. I will describe the experimental strategy behind “single‐molecule sorting”, as well as its application to the analysis of several DNA repair helicases, which produce transient single‐stranded intermediates of DNA replication, recombination and repair. Individual protein molecules isolated from human cells with their native PTMs are analyzed using total internal reflection fluorescence microscopy (TIRFM). Separation of the activity trajectories originated from modified and unmodified enzymes allows us to quantify the fraction of the protein modified in the cell and to assess the effect of each modification or a combination of modifications on the target activity.
Details
- Title: Subtitle
- Single molecule sorting of the DNA repair machines (477.1)
- Creators
- Maria Spies - University of Iowa
- Resource Type
- Abstract
- Publication Details
- The FASEB journal, Vol.28(S1), p.n/a
- Publisher
- The Federation of American Societies for Experimental Biology
- DOI
- 10.1096/fasebj.28.1_supplement.477.1
- ISSN
- 0892-6638
- eISSN
- 1530-6860
- Number of pages
- 1
- Language
- English
- Date published
- 04/2014
- Academic Unit
- Radiation Oncology; Biochemistry and Molecular Biology
- Record Identifier
- 9984293413102771
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