RESUMO
Homologous recombination (HR) helps maintain genome integrity, and HR defects give rise to disease, especially cancer. During HR, damaged DNA must be aligned with an undamaged template through a process referred to as the homology search. Despite decades of study, key aspects of this search remain undefined. Here, we use single-molecule imaging to demonstrate that Rad54, a conserved Snf2-like protein found in all eukaryotes, switches the search from the diffusion-based pathways characteristic of the basal HR machinery to an active process in which DNA sequences are aligned via an ATP-dependent molecular motor-driven mechanism. We further demonstrate that Rad54 disrupts the donor template strands, enabling the search to take place within a migrating DNA bubble-like structure that is bound by replication protein A (RPA). Our results reveal that Rad54, working together with RPA, fundamentally alters how DNA sequences are aligned during HR.
Assuntos
Trifosfato de Adenosina/genética , DNA Helicases/genética , Enzimas Reparadoras do DNA/genética , DNA/genética , Recombinação Homóloga/genética , Proteínas de Saccharomyces cerevisiae/genética , Adenosina Trifosfatases/genética , Dano ao DNA/genética , Reparo do DNA/genética , Proteínas de Ligação a DNA/genética , Hidrólise , Saccharomyces cerevisiae/genética , Alinhamento de Sequência/métodosRESUMO
In the repair of DNA double-strand breaks by homologous recombination, the DNA break ends must first be processed into 3' single-strand DNA overhangs. In budding yeast, end processing requires the helicase Sgs1 (BLM in humans), the nuclease/helicase Dna2, Top3-Rmi1, and replication protein A (RPA). Here, we use single-molecule imaging to visualize Sgs1-dependent end processing in real-time. We show that Sgs1 is recruited to DNA ends through Top3-Rmi1-dependent or -independent means, and in both cases Sgs1 is maintained in an immoble state at the DNA ends. Importantly, the addition of Dna2 triggers processive Sgs1 translocation, but DNA resection only occurs when RPA is also present. We also demonstrate that the Sgs1-Dna2-Top3-Rmi1-RPA ensemble can efficiently disrupt nucleosomes, and that Sgs1 itself possesses nucleosome remodeling activity. Together, these results shed light on the regulatory interplay among conserved protein factors that mediate the nucleolytic processing of DNA ends in preparation for homologous recombination-mediated chromosome damage repair.
Assuntos
Quebras de DNA de Cadeia Dupla , DNA Helicases/metabolismo , Reparo do DNA , RecQ Helicases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Recombinação Homóloga , Nucleossomos/metabolismo , Proteína de Replicação A/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Imagem Individual de Molécula/métodosRESUMO
Ngo et al. use single-molecule methods to show that DNA can be more readily displaced from one side of a nucleosome relative to the other side. This unexpected mechanical asymmetry may offer a path of least resistance, allowing RNA polymerases to traverse nucleosomes if they approach from the correct direction.
