title: Mechanisms by which Bloom protein can disrupt recombination intermediates of Okazaki fragment maturation.
abstract: Bloom syndrome is a familial genetic disorder associated with sunlight sensitivity and a high predisposition to cancers. The mutated gene, Bloom protein (BLM), encodes a DNA helicase that functions in genome maintenance via roles in recombination repair and resolution of recombination structures. We designed substrates representing illegitimate recombination intermediates formed when a displaced DNA flap generated during maturation of Okazaki fragments escapes cleavage by flap endonuclease-1 and anneals to a complementary ectopic DNA site. Results show that displaced, replication protein A (RPA)-coated flaps could readily bind and ligate at the complementary site to initiate recombination. RPA also displayed a strand-annealing activity that hastens the rate of recombination intermediate formation. BLM helicase activity could directly disrupt annealing at the ectopic site and promote flap endonuclease-1 cleavage. Additionally, BLM has its own strand-annealing and strand-exchange activities. RPA inhibited the BLM strand-annealing activity, thereby promoting helicase activity and complex dissolution. BLM strand exchange could readily dissociate invading flaps, e.g. in a D-loop, if the exchange step did not involve annealing of RPA-coated strands. Use of ATP to activate the helicase function did not aid flap displacement by exchange, suggesting that this is a helicase-independent mechanism of complex dissociation. When RPA could bind, it displayed its own strand-exchange activity. We interpret these results to explain how BLM is well equipped to deal with alternative recombination intermediate structures.
authors.affiliation: Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, 601 Elmwood Avenue, Rochester, NY 14642, USA.
authors.forename: Jeremy D
authors.initials: JD
authors.lastname: Bartos
authors.forename: Wensheng
authors.initials: W
authors.lastname: Wang
authors.forename: Jason E
authors.initials: JE
authors.lastname: Pike
authors.forename: Robert A
authors.initials: RA
authors.lastname: Bambara
mesh_terms: D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism*; D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism*; D004260:DNA Repair; D045585:Flap Endonucleases / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D009690:Nucleic Acid Conformation; D053484:RecQ Helicases; D011995:Recombination, Genetic; D051716:Replication Protein A / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: C022262:Okazaki fragments; D051716:Replication Protein A; D004247:DNA; D045585:Flap Endonucleases; D000251:Adenosine Triphosphatases; C097151:Bloom syndrome protein; D004265:DNA Helicases; D053484:RecQ Helicases