DNA end resection by Dna2–Sgs1–RPA and its stimulation by Top3–Rmi1 and Mre11–Rad50–Xrs2

The repair of DNA double-strand breaks (DSBs) by homologous recombination requires processing of broken ends. For repair to start, the DSB must first be resected to generate a 3′-single-stranded DNA (ssDNA) overhang, which becomes a substrate for the DNA strand exchange protein, Rad51 (ref. 1). Genetic studies have implicated a multitude of proteins in the process, including helicases, nucleases and topoisomerases. Here we biochemically reconstitute elements of the resection process and reveal that it requires the nuclease Dna2, the RecQ-family helicase Sgs1 and the ssDNA-binding protein replication protein-A (RPA). We establish that Dna2, Sgs1 and RPA constitute a minimal protein complex capable of DNA resection in vitro. Sgs1 helicase unwinds the DNA to produce an intermediate that is digested by Dna2, and RPA stimulates DNA unwinding by Sgs1 in a species-specific manner. Interestingly, RPA is also required both to direct Dna2 nucleolytic activity to the 5′-terminated strand of the DNA break and to inhibit 3′ to 5′ degradation by Dna2, actions that generate and protect the 3′-ssDNA overhang, respectively. In addition to this core machinery, we establish that both the topoisomerase 3 (Top3) and Rmi1 complex and the Mre11–Rad50–Xrs2 complex (MRX) have important roles as stimulatory components. Stimulation of end resection by the Top3–Rmi1 heterodimer and the MRX proteins is by complex formation with Sgs1 (refs 5, 6), which unexpectedly stimulates DNA unwinding. We suggest that Top3–Rmi1 and MRX are important for recruitment of the Sgs1–Dna2 complex to DSBs. Our experiments provide a mechanistic framework for understanding the initial steps of recombinational DNA repair in eukaryotes

Experimental Verification of a New Model of Acoustic Oscillations in Supersonic Cavity Flows

In this paper, we experimentally verify a new model of the acoustic oscillation that is observed in supersonic flows over a rectangular cavity. This model was developed previously in order to understand why the jump in the predominant oscillation frequency occurred when the length-to-depth ratio of the cavity was gradually varied. In the present experiment, the target flows are supersonic flows over a rectangular cavity of variable length-to-depth ratios. The inlet Mach number is selected to be 1.65. The length-to-depth ratio of the cavity is varied by changing the depth as the length remains constant. The pressure oscillation is measured by the semiconductor type pressure sensor mounted on the wall downstream of the trailing edge of the cavity, and the predominant frequencies are obtained experimentally for each length-to-depth ratio. Also, the predominant frequencies are calculated by using the model. The results obtained by using the model are found to agree with the experimental results with respect to both the frequency jump and the variation in frequency with the length-to-depth ratio