Mechanism of the ATP-dependent DNA end-resection machinery from Saccharomyces cerevisiae

Abstract

If not properly processed and repaired, DNA double-strand breaks (DSBs) can give rise to deleterious chromosome rearrangements, which could ultimately lead to the tumor phenotype 1, 2. DSB ends are resected in a 5′ to 3′ fashion in cells, to yield single-stranded DNA for the recruitment of factors critical for DNA damage checkpoint activation and repair by homologous recombination2. The resection process involves redundant pathways consisting of nucleases, DNA helicases, and associated proteins3. Being guided by recent genetic studies 4-6 , we have reconstituted the first eukaryotic ATP-dependent DNA end resection machinery comprising the Saccharomyces cerevisiae Mre11-Rad50-Xrs2 (MRX) complex, the Sgs1-Top3-Rmi1 (STR) complex, Dna2 protein and the heterotrimeric single-strand DNA binding protein RPA. We show that DNA strand separation during end resection is mediated by the Sgs1 helicase function, in a manner that is enhanced by Top3-Rmi1 and MRX. In congruence with genetic observations 6 , while the Dna2 nuclease activity is critical for resection, the Mre11 nuclease activity is dispensable. By examining the top3 Y356F allele and its encoded protein, we provide evidence that the topoisomerase activity of Top3, although critical for the suppression of crossover recombination 2,7 , is not needed for resection either in cells or in the reconstituted system. Our results also unveil a multi-faceted role of RPA, in the sequestration of ssDNA generated by DNA unwinding, enhancement of 5′ strand incision, and protection of the 3′ strand. Our reconstituted system should serve as a useful model for delineating the mechanistic intricacy of the DNA break resection process in eukaryotes.The 3′ ssDNA strands derived from DSB resection attract RPA, which promotes the recruitment of checkpoint proteins to effect cell cycle arrest 8 . With the aid of a recombination mediator protein, such as yeast Rad52 or human BRCA2, the Rad51 recombinase displaces RPA from the ssDNA to assemble into a right-handed helical polymer capable of initiating DSB repair by homologous recombination 1,2 . Genetic studies in yeast have shown that DSB resection proceeds in two steps. The MRX complex plays a 3 To whom correspondences and request for materials should be addressed: Gregory Ira: gira@bcm.edu, Patrick Sung: patrick.sung@yale.edu. role in initiation, while the Sgs1 helicase, its associated proteins Top3 and Rmi1, and the helicase/nuclease Dna2, whose nuclease activity is needed for Okazaki fragment processing 9,10 , constitute the DNA motor-driven path of long-range resection. Exo1, a 5′-3′ exonuclease, defines a redundant resection means 4-6 . Here we reconstitute the Sgs1/Dna2-dependent DNA resection machinery and present results germane for understanding its mechanistic underpinnings.The requisite factors, namely, Sgs1, Top3-Rmi1 (TR) complex, MRX complex, Dna2, and RPA were purified and analyzed (see Supplementary Fig. 2 and the Supplementary Information). As shown in Figure 1a, the combination of these factors degraded a 1.9-kb ...

Single-molecule imaging reveals the mechanism of Exo1 regulation by single-stranded DNA binding proteins

“…Because hExo1 is the major nuclease in human DSB resection (11), hExo1 knockdown severely limits DNA resection intermediates (12). A second, partially redundant resection pathway requires DNA2 nuclease (19,20,22,23). To specifically monitor hExo1-dependent resection, we first measured resection in DNA2-depleted cells.…”

“…RPA protects the ssDNA from degradation, participates in DNA damage response signaling, and acts as a loading platform for downstream DSB repair proteins (15)(16)(17). RPA also coordinates DNA resection by removing secondary ssDNA structures and by modulating the Bloom syndrome, RecQ helicase-like (BLM)/ DNA2-and Exo1-dependent DNA resection pathways (18)(19)(20)(21). Reconstitution of both the yeast and human BLM (Sgs1 in yeast)/DNA2-dependent resection reactions established that RPA stimulates DNA unwinding by BLM/Sgs1 and enforces a 5′-endonuclease polarity on DNA2 (20,22).…”

Microhomology-mediated End Joining and Homologous Recombination share the initial end resection step to repair DNA double-strand breaks in mammalian cells

“…In yeast, Mre11 interacts with and recruits Sgs1 to DSB ends (46,47). In mammalian cells, the physical interaction of MRN with BLM/Exo1 may also promote binding of BLM and Exo1 to DSB ends and facilitate end resection (48).…”

Roles of DNA helicases and Exo1 in the avoidance of mutations induced by Top1-mediated cleavage at ribonucleotides in DNA

“…1,[19][20][21] For helicase and nuclease assays, a duplex DNA substrate with a unique U residue was treated by topoisomerase I from calf thymus (Invitrogen) to create a nick at the ribonucleoside site. 1 Helicase, nuclease, and affinity pull-down assays were conducted as before.…”