Replication Protein A Enhances Kinetics of Uracil DNA Glycosylase on ssDNA and Across DNA Junctions: Explored with a DNA Repair Complex Produced with SpyCatcher/SpyTag Ligation |
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Authors: | Sharon N Greenwood Dr Rashmi S Kulkarni Dr Michel Mikhail Dr Brian P Weiser |
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Affiliation: | 1. Department of Molecular Biology, Rowan University School of Osteopathic Medicine, Stratford, NJ 08084 USA;2. Department of Molecular Biology, Rowan University School of Osteopathic Medicine, Stratford, NJ 08084 USA
Department of Internal Medicine, Newark Beth Israel Medical Center, Newark, NJ 07112 USA |
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Abstract: | DNA repair proteins participate in extensive protein?protein interactions that promote the formation of DNA repair complexes. To understand how complex formation affects protein function during base excision repair, we used SpyCatcher/SpyTag ligation to produce a covalent complex between human uracil DNA glycosylase (UNG2) and replication protein A (RPA). Our covalent “RPA?Spy?UNG2” complex could identify and excise uracil bases in duplex areas next to ssDNA?dsDNA junctions slightly faster than the wild-type proteins, but this was highly dependent on DNA structure, as the turnover of the RPA?Spy?UNG2 complex slowed at DNA junctions where RPA tightly engaged long ssDNA sections. Conversely, the enzymes preferred uracil sites in ssDNA where RPA strongly enhanced uracil excision by UNG2 regardless of ssDNA length. Finally, RPA was found to promote UNG2 excision of two uracil sites positioned across a ssDNA?dsDNA junction, and dissociation of UNG2 from RPA enhanced this process. Our approach of ligating together RPA and UNG2 to reveal how complex formation affects enzyme function could be applied to examine other assemblies of DNA repair proteins. |
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Keywords: | enzyme RPA spycatcher UNG2 uracil |
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