Dual DNA-binding domains shape the interaction of Brh2 with DNA |
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| Affiliation: | 1. Department of Chemistry, College of Staten Island, City University of New York, 2800 Victory Boulevard, Staten Island, NY 10314, USA;2. Ph.D. Program in Chemistry, The Graduate Center of the City University of New York, 365 Fifth Avenue, New York, NY 10016, USA;3. ExxonMobil Research and Engineering Company, 1545 Rt 22 East, Annandale, NJ 08801, USA;1. Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210, USA;2. The Ohio State Biophysics Program, The Ohio State University, Columbus, OH 43210, USA;1. Department of Chemistry and Biochemistry, Clippinger Laboratories, Ohio University, Athens, OH 45701, United States;2. Department of Chemistry, Knight Chemical Laboratory, University of Akron, Akron, OH 44325, United States;1. Postdoctoral Research Station of Basic Medicine & Hengyang Medical School & Hunan Province Cooperative Innovation Center for Molecular Target New Drug Study & School of Pharmaceutical Science, University of South China, Hengyang, Hunan 421001, China;2. State Key Laboratory of Applied Organic Chemistry & Key Laboratory of Nonferrous Metal Chemistry and Resources Utilization of Gansu Province & Department College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou, Gansu 730000, China |
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| Abstract: | Brh2, the BRCA2 ortholog in the fungus Ustilago maydis, harbors two different DNA-binding domains, one located in the N-terminal region and the other located in the C-terminal region. Here we were interested in comparing the biochemical properties of Brh2 fragments, Brh2NT and Brh2CT, respectively, harboring the two different DNA-binding regions to understand the mechanistic purpose of dual DNA-interaction domains. With oligonucleotide substrates to model different DNA conformations, it was found that the substrate specificity of Brh2NT and Brh2CT was almost indistinguishable although avidity was different depending on salt concentration. DNA annealing activity inherent in Brh2 was found to be attributable to Brh2NT. Likewise, activity responsible for a second-end capture reaction modeling a later step in repair of DNA double-strand breaks was found attributable to Brh2NT. Efficient annealing of DNA strands coated with RPA required full length Brh2 rather than Brh2NT suggesting Brh2CT contributes to the activity when RPA is present. Brh2NT and Brh2CT were both found capable of physically interacting with RPA. The results suggest that while the two DNA-binding regions of Brh2 appear functionally redundant in certain aspects of DNA repair, they differ in fundamental properties, and likely contribute in different ways to repair processes involving or arising from stalled DNA replication forks. |
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| Keywords: | BRCA2 Rad51 Homologous recombination DNA repair DNA binding DNA annealing Second-end capture |
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