Browsing by Subject "Sae2"
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Item Characterization of Mre11/Rad50/Xrs2, Sae2, and Exo1 in DNA end resection(2008-08) Nicolette, Matthew Lawrence; Paull, Tanya T.Eukaryotic cells repair DNA double-strand breaks (DSBs) through both non-homologous and homologous recombination pathways. The initiation of homologous recombination requires the generation of 3' overhangs, which are essential for the formation of Rad51 protein-DNA filaments that catalyze subsequent steps of strand invasion. Experiments in budding yeast show that resection of the 5' strand at a DSB is delayed in strains lacking any components of the Mre11/Rad50/Xrs2 (MRX) complex¹ . In meiosis, a specific class of hypomorphic mutants of mre11 and rad50 (Rad50S) are completely deficient in 5' resection and leave Spo11 covalently attached to the 5' strands of DNA breaks². Similar to mre11S and rad50S mutants, sae2 deletion strains fail to resect 5' strands at meiotic DSBs and accumulate covalent Spo11 adducts³;⁴. In addition, Sae2 and MRX were also found to function cooperatively to process hairpin-capped DNA ends in vivo in yeast. sae2 and mrx null strains show a severe defect in processing these structures and accumulate hairpin-capped DNA ends⁵;⁶. The Longhese laboratory has also shown that Sae2 deletion strains show a delay in 5' strand resection, similar to rad50S strains⁷. Recently, Bettina Lengsfeld in our laboratory demonstrated that Sae2 itself possesses nuclease activity and that MRX and Sae2 act cooperatively to cleave single-stranded DNA adjacent to DNA hairpin structures⁸. In vitro characterization of Sae2 showed that the central and N-terminal domains are required for MRX-independent nuclease activity and that the C-terminus is required for cooperative activities with MRX. Sae2 also acts independently of MRX as a 5' flap endonuclease on branched structures in vitro. Our studies investigate whether MRX, Sae2, and Exo1 function cooperatively in DNA resection using recombinant, purified proteins in vitro. We developed assays utilizing strand-specific Southern blot analysis to visualize DNA end processing of model DNA substrates using recombinant proteins in vitro. Our results demonstrate that MRX and Sae2 cooperatively resect the 5' end of a DNA duplex together with the Exo1 enzyme, supporting a role for these factors in the early stages of homologous recombination and repair.Item Characterization of the novel endonuclease Sae2 involved in DNA end processing(2011-08) Shen, Mingjuan; Paull, Tanya T.; Iyer, Vishwanath R.; Jayaram, Makkuni; Johnson, Arlen W.; Shen, XuetongAt the very center of sexual reproduction is meiosis. During meiosis, the formation of meiotic Double-Strand-Breaks (DBSs) and their repair by homologous recombination are widely conserved events occurring among most eukaryote species. Meiosis-specific DSB formation requires at least nine proteins (Spo11, Ski8, Rec102, Rec104, Mei4, Mer2, Rec114, Mre11/Rad50/Xrs2) in S. cerevisiae, and the resection of the DSB ends requires additional four proteins (Mre11/Rad50/Xrs2, and Sae2). Spo11 has been identified as the catalytic component of this DSB-initiating complex. However, the roles played by the majority of these proteins are not clear. I have purified the recombinant Spo11/Ski8/Rec102/Rec104 complex, characterized its DNA binding ability as well as its cleavage activity on supercoiled plasmid DNA. Sae2 functions in both meiotic and mitotic repair of DNA double-strand breaks (DSBs) in S. cerevisiae. In vivo experiments have shown that Sae2 collaborates with the Mre11/Rad50/Xrs2 (MRX) complex in DNA end processing. Our laboratory previously showed that recombinant Sae2 exhibits endonuclease activity on single-stranded DNA and single-strand/double-strand DNA junctions using purified proteins in vitro. The MRX complex stimulates Sae2 endonuclease activity on single-stranded DNA close to single-strand/double-strand junctions, through its endonucleolytic activity. However, Sae2 contains no conserved typical nuclease domain, and it only shares very limited homology with its human functional counterpart CtIP. To characterize Sae2 and the active sites responsible for its nuclease activity, I used partial proteolysis and site-directed mutagenesis to analyze the protein. Biochemical assays in vitro show that acidic residues in the central domain play an important role in Sae2 endonuclease activity. Sae2 has also been shown to be phosphorylated by CDK (Cyclin-Dependent Kinase) during the S and G2 phases of the cell cycle, as well as by Tel1/Mec1 upon DNA damage. These modifications are essential for the function of Sae2 in DNA repair, but the function of these modifications are not clear. I have demonstrated that, in the presence of MRX, Sae2 (5D/S267E) mimicking constitutive phosphorylation by CDK and Mec1/Tel1 can assist the 5’ to 3’ exonuclease Exo1 significantly in 5’ end resection by suppressing the inhibitory effect of Ku. These results suggest that Sae2 is a critical switching protein which determines the choice between HR and NHEJ in yeast cells upon DNA damage.Item Regulation of the activity of a budding yeast DNA damage repair enzyme Sae2(2014-12) Fu, Qiong, Ph. D.; Paull, Tanya T.; Iyer, Vishwanath R; Jayaram, Makkuni; Johnson, Arlen W; Zhang, YanIn response to DNA damage, many repair and signaling molecules mobilize rapidly to the sites of DNA double-strand breaks (DSBs). This network of immediate responses is regulated at the level of post-translational modifications to coordinate DNA repair and checkpoint signaling. Here we investigate the DNA damage-induced oligomeric transitions of the Sae2 protein, an important enzyme in the initiation of DSB repair. Sae2 is a target of multiple phosphorylation events, which we identify and characterize in vivo in budding yeast. Both cell cycle-dependent and DNA damage-induced phosphorylation of Sae2 are important for the cell survival after DNA damage, and the cell cycle-regulated modifications are required to prime the damage-dependent events. We find that Sae2 exists in the form of inactive oligomers that are transiently released into smaller active units by these series of phosphorylation events. DNA damage also triggers removal of Sae2 through autophagy and proteasomal degradation, ensuring that active Sae2 is present only transiently in cells. This analysis provides evidence for a novel type of protein regulation where the activity of an enzyme is controlled dynamically by post-translational modifications that regulate its solubility and oligomeric state. Budding yeast Ess1 is a phosphorylation-specific prolyl isomerase. Its human homolog Pin1 is found to isomerize CtIP, the human functional ortholog of Sae2, and promote the proteasomal degradation of CtIP. However, I could neither detect any interaction between Ess1 and Sae2, nor observe any change in Sae2 protein level while overexpressing wild-type or mutant Ess1, suggesting Ess1 does not act on Sae2, like Pin1 does on CtIP. The increased DNA damage sensitivity of Ess1 mutants indicates that Ess1 is involved in DNA repair, but not related to Sae2. Since Ess1 plays an important role in transcription termination together with a RNA 3’ end processing factor Pcf11, I overexpressed wild-type Pcf11 and found it significantly increased the DNA damage resistance of either wild-type or H164R mutant Ess1 cells, and also the sae2Δ cells. These results imply that Ess1, Pcf11 and Sae2 might contribute to DNA damage repair through transcription termination, which links transcription termination and DNA damage repair together.Item Role of Sae2 in repair of TopoisomeraseI-DNA conjugates(2016-06-17) Arora, Sucheta; Paull, Tanya T.; Johnson, Arlen W; Huibregtse, Jon M; Vasquez, Karen M; Jayaram, MakkuniTopoisomerase inhibitors are widely used chemotherapeutic drugs. They covalently trap the target topoisomerase on DNA, which interferes with the progression of replication and transcription machinery and induces lethal DNA damage. The ability of cells to repair this damage is a key determinant of the effectiveness of these drugs and elucidation of the repair pathways is of great clinical relevance. In this study, I investigate the role of Sae2 protein in protecting yeast cells from camptothecin, a Topoisomerase I inhibitor. Sae2 is a key component of the DNA double strand break repair pathway and works in collaboration with the Mre11-Rad50-Xrs2 (MRX) protein complex in homologous recombination mediated repair. Sae2 null cells are highly sensitive to camptothecin in survival assays. I show that this sensitivity is dependent on active replication as well as transcription in vivo. Sae2 preferentially localizes to highly transcribed regions after camptothecin exposure in S phase, suggesting that it has a role in repair of TopoI-DNA conjugates at those sites. We find that camptothecin sensitivity in the absence of Sae2 can be rescued by over-expression of Sen1, an RNA:DNA helicase that is part of the transcription termination NRD complex. Sen1 removes R-loops that form in the negatively supercoiled region behind a stalled or slow transcription unit. Sen1 over-expression also rescues a strain containing both a sae2 null allele and an Mre11 nuclease-deficient allele. I show that R-loops accumulate at the highly transcribed rDNA region after camptothecin exposure in the absence of Sae2 function and that Sen1 over-expression reduces this accumulation. I propose that Sae2 and Mre11 have a role in processing the R loops induced by TopoI-DNA conjugates at highly transcribed regions in the genome. It has been shown that Sae2 is a structure-specific endonuclease with a preference for 5’ flaps and ssDNA/dsDNA junctions in vitro. It also stimulates the nuclease activity of Mre11 on DNA ends containing protein adducts. In this study, I identify two mutant alleles of Sae2 that separate these two biochemical activities. The D285P K288P Sae2 mutant is proficient in the Sae2 nuclease activity but deficient for the stimulation of Mre11 activity on protein-blocked DNA ends. The E161P K163P mutation renders the protein deficient for its nuclease activity but the mutant can stimulate Mre11 activity. The survival complementation assay indicates that stimulation of Mre11 activity is the primary function of Sae2 and that its nuclease activity is required only in the absence of Mre11 nuclease activity. Sae2 is implicated in a variety of roles in DNA double-strand break repair and signaling pathways including stimulating resection, modulating Tel1 activation, removing MRX from the DSB sites, processing hairpin intermediates, removing Spo11 from meiotic DNA breaks and processing Topo1-DNA adducts. The mutant alleles described in this study provide useful reagents to elucidate the role of Sae2 enzymatic activity and its coordination with Mre11 nuclease activity in different biological functions.