D-loop formation by Brh2 protein of Ustilago maydis.
Brh2, the ortholog of the BRCA2 tumor suppressor in Ustilago maydis, works hand in hand with Rad51 to promote repair of DNA by homologous recombination. Previous studies established that Brh2 can stimulate DNA strand exchange by enabling Rad51 nucleoprotein filament formation on replication protein A-coated ssDNA. But, more recently, it was noted that Brh2 has an inherent DNA annealing activity, raising the notion that it might have roles in recombination in addition to or beyond the mediator function. Here, we found that Brh2 can autonomously promote the formation of D-loops in reactions with plasmid DNA and homologous single-stranded oligonucleotides. The reaction differs from that catalyzed by Rad51 in having no requirement for cofactors or preloading phase on ssDNA. D-loop formation was most effective when Brh2 was mixed with plasmid DNA before addition of single-stranded oligomer. D-loop formation catalyzed by Rad51 was also enhanced when Brh2 was premixed with plasmid DNA. Brh2 rendered defective in Rad51 interaction by mutation in the BRC element was still capable of promoting D-loop formation. However, the mutant protein was unable to enhance the Rad51-catalyzed reaction. The results suggest a model in which Brh2 binding to plasmid DNA attracts and helps capture Rad51-coated ssDNA.
Mazloum
Nayef
N
Department of Microbiology and Immunology, Cornell University Weill Medical College, New York, NY 10065, USA.
Zhou
Qingwen
Q
Holloman
William K
WK
D024682:BRCA2 Protein / Q000737:chemistry / Q000502:physiology*; D004247:DNA / Q000737:chemistry; D004277:DNA, Single-Stranded / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000502:physiology; D004926:Escherichia coli / Q000378:metabolism; D005095:Exoribonucleases; D005786:Gene Expression Regulation; D008968:Molecular Conformation; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D051135:Rad51 Recombinase / Q000737:chemistry; D011995:Recombination, Genetic; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry; D014588:Ustilago
D024682:BRCA2 Protein; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D029701:Saccharomyces cerevisiae Proteins; D004247:DNA; D051135:Rad51 Recombinase; D005095:Exoribonucleases; C098317:DSS1 protein, S cerevisiae