title: Dual DNA-binding domains shape the interaction of Brh2 with DNA.
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, Brh2(NT) and Brh2(CT), 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 Brh2(NT) and Brh2(CT) was almost indistinguishable although avidity was different depending on salt concentration. DNA annealing activity inherent in Brh2 was found to be attributable to Brh2(NT). Likewise, activity responsible for a second-end capture reaction modeling a later step in repair of DNA double-strand breaks was found attributable to Brh2(NT). Efficient annealing of DNA strands coated with RPA required full length Brh2 rather than Brh2(NT) suggesting Brh2(CT) contributes to the activity when RPA is present. Brh2(NT) and Brh2(CT) 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.
authors.affiliation: Department of Microbiology and Immunology, and Weill Cornell Cancer Center, Weill Cornell Medical College, New York, NY 10065, United States.
authors.forename: Qingwen
authors.initials: Q
authors.lastname: Zhou
authors.affiliation: Department of Microbiology and Immunology, and Weill Cornell Cancer Center, Weill Cornell Medical College, New York, NY 10065, United States. Electronic address: wkhollo@med.cornell.edu.
authors.forename: William K
authors.initials: WK
authors.lastname: Holloman
mesh_terms: D024682:BRCA2 Protein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004260:DNA Repair; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism*; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009841:Oligonucleotides / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051716:Replication Protein A / Q000378:metabolism; D014588:Ustilago / Q000201:enzymology* / Q000235:genetics
chemical_list: D024682:BRCA2 Protein; D004277:DNA, Single-Stranded; D005656:Fungal Proteins; D009841:Oligonucleotides; D051716:Replication Protein A
keywords: BRCA2; DNA annealing; DNA binding; DNA repair; Homologous recombination; Rad51; Second-end capture