Identification of Nucleases and Phosphatases by Direct Biochemical Screen of the Saccharomyces cerevisiae Proteome

The availability of yeast strain collections expressing individually tagged proteins to facilitate one-step purification provides a powerful approach to identify proteins with particular biochemical activities. To identify novel exo- and endo-nucleases that might function in DNA repair, we undertook a proteomic screen making use of the movable ORF (MORF) library of yeast expression plasmids. This library consists of 5,854 yeast strains each expressing a unique yeast ORF fused to a tripartite tag consisting of His6, an HA epitope, a protease 3C cleavage site, and the IgG-binding domain (ZZ) from protein A, under the control of the GAL1 promoter for inducible expression. Pools of proteins were partially purified on IgG sepharose and tested for nuclease activity using three different radiolabeled DNA substrates. Several known nucleases and phosphatases were identified, as well as two new members of the histidine phosphatase superfamily, which includes phosphoglycerate mutases and phosphatases. Subsequent characterization revealed YDR051c/Det1 to be an acid phosphatase with broad substrate specificity, whereas YOR283w has a broad pH range and hydrolyzes hydrophilic phosphorylated substrates. Although no new nuclease activities were identified from this screen, we did find phosphatase activity associated with a protein of unknown function, YOR283w, and with the recently characterized protein Det1. This knowledge should guide further genetic and biochemical characterization of these proteins

Phosphatase activities of Det1 and YOR283w.

<p>A. Purification of GST-YOR283w. Proteins were separated on a 12% SDS-polyacrylamide gel and stained with Coomassie blue. Lane M, molecular mass markers (kDa); lane 1, induced cell lysate; lanes 2 and 3, elution from glutathione agarose with 25 mM reduced glutathione buffer. Position of the GST-YOR283w fusion protein is indicated. Phosphatase activity of YOR283w using pH 7.7 buffer detected by TLC (B) or Det 1 at pH 4.4 or pH 7.7 (C). Reaction mixtures contain 250 ng GST-YOR283w, 5 µl aliquot of IgG sepharose-bound Det1 recombinant library protein or 1 unit of CIP (control), pH 4.4 or 7.7 buffers (50 mM) as indicated, supplemented with or without 1/100 phosphatase inhibitor (PI) cocktail set II (Calbiochem). Reaction mixtures were incubated at 37°C for 1 hr and reaction products were resolved by PEI-cellulose TLC plates. Positions of ATP and P_i are indicated.</p

Characterization of the Det1 and YOR283w phosphatase activities.

<p>Optimum pH (A) metal dependence (B) and substrate specificity (C) of Det1 and YOR283w were measured by the release of inorganic phosphate as described in Experimental Procedures. σ, sodium acetate/acetic acid buffer; ⧫, Tris-acetate or Tris-HCl buffer; ν, CAPS/NaOH buffer.</p

Substrates to assay for nuclease, phosphatase and helicase activities.

<p>A. Phosphatases can catalyze the removal of ³²P_i from ssDNA; 5′ to 3′ single-strand specific exonucleases can degrade DNA to release ³²P-labeled nucleotides, 3′ to 5′ single-strand specific exonucleases degrade the 3′ end resulting in a shortened labeled substrate, or the substrate can be cleaved by endonucleases to generate ³²P-labeled DNA fragments. B. Structure-specific endonucleases can cleave the single-stranded DNA tail adjacent to duplex region of the Y DNA substrate. In the presence of ATP, helicases can unwind Y substrate to the two constituent ssDNA oligonucleotides. C. Holliday junctions can be cleaved by a resolvase to generate nicked duplex products by introducing paired incisions across the junction.</p

Identification of nucleases and a helicase from the MORF library.

<p>Reaction mixtures contained an aliquot of the indicated protein pool or purified from an individual library strain incubated with one of the three radiolabeled DNA substrates (ssDNA, Y or HJ). A. The Y substrate was incubated with T7 exonuclease (control) or three different protein pools (60–62). The activity in pool 61 is due to Rad27. B. The HJ substrate was incubated with <i>E. coli</i> RuvC (control) or seven different protein pools (103–109). Pool 109 contains Cce1. C. The Y substrate was incubated with four different protein pools (120–123). The activity in pool 122 is due to Nam7. D. The ssDNA was incubated with <i>E. coli</i> RecJ (control), Trm10, Rai1, or other proteins purified from individual library strains (not indicated). Reaction products were resolved by 10% (for Y and HJ) or 15% (for ssDNA) native polyacrylamide gels and analyzed using a phosphorimager. Positions of reaction substrates and products after gel electrophoresis are indicated.</p

Identification of phosphatases.

<p>Reaction mixtures contained ssDNA substrate and an aliquot of the indicated protein pool (224–226, 44–46 and 132–134) or <i>E. coli</i> RecJ (control). Protein pool 225, 45 and 132 contained Pho8, Det1 and YOR283w library proteins, respectively. Reaction products were resolved by 15% native polyacrylamide gels and analyzed using a phosphorimager. Positions of ssDNA substrate, released ³²P-dNTP and ³²P_i after gel electrophoresis are indicated.</p

Coupling of Homologous Recombination and the Checkpoint by ATR

ATR is a key regulator of cell-cycle checkpoints and homologous recombination (HR). Paradoxically, ATR inhibits CDKs during checkpoint responses, but CDK activity is required for efficient HR. Here, we show that ATR promotes HR after CDK-driven DNA end resection. ATR stimulates the BRCA1-PALB2 interaction after DNA damage and promotes PALB2 localization to DNA damage sites. ATR enhances BRCA1-PALB2 binding at least in part by inhibiting&nbsp;CDKs. The optimal interaction of BRCA1 and PALB2 requires phosphorylation of PALB2 at S59, an ATR site, and hypo-phosphorylation of S64, a CDK site. The PALB2-S59A/S64E mutant is defective for localization to DNA damage sites and HR, whereas the PALB2-S59E/S64A mutant partially bypasses ATR for its localization. Thus, HR is a biphasic process requiring both high-CDK and low-CDK periods. As exemplified by the regulation of PALB2 by ATR, ATR promotes HR by orchestrating a "CDK-to-ATR switch" post-resection, directly coupling the checkpoint to HR

ATR inhibition disrupts rewired homologous recombination and fork protection pathways in PARP inhibitor-resistant BRCA-deficient cancer cells

Poly-(ADP-ribose) polymerase (PARP) inhibitors (PARPis) selectively kill BRCA1/2-deficient cells, but their efficacy in BRCA-deficient patients is limited by drug resistance. Here, we used derived cell lines and cells from patients to investigate how to overcome PARPi resistance. We found that the functions of BRCA1 in homologous recombination (HR) and replication fork protection are sequentially bypassed during the acquisition of PARPi resistance. Despite the lack of BRCA1, PARPi-resistant cells regain RAD51 loading to DNA double-stranded breaks (DSBs) and stalled replication forks, enabling two distinct mechanisms of PARPi resistance. Compared with BRCA1-proficient cells, PARPi-resistant BRCA1-deficient cells are increasingly dependent on ATR for survival. ATR inhibitors (ATRis) disrupt BRCA1-independent RAD51 loading to DSBs and stalled forks in PARPi-resistant BRCA1-deficient cells, overcoming both resistance mechanisms. In tumor cells derived from patients, ATRis also overcome the bypass of BRCA1/2 in fork protection. Thus, ATR inhibition is a unique strategy to overcome the PARPi resistance of BRCA-deficient cancers

ATR inhibition disrupts rewired homologous recombination and fork protection pathways in PARP inhibitor-resistant BRCA-deficient cancer cells

Poly-(ADP-ribose) polymerase (PARP) inhibitors (PARPis) selectively kill BRCA1/2-deficient cells, but their efficacy in BRCA-deficient patients is limited by drug resistance. Here, we used derived cell lines and cells from patients to investigate how to overcome PARPi resistance. We found that the functions of BRCA1 in homologous recombination (HR) and replication fork protection are sequentially bypassed during the acquisition of PARPi resistance. Despite the lack of BRCA1, PARPi-resistant cells regain RAD51 loading to DNA double-stranded breaks (DSBs) and stalled replication forks, enabling two distinct mechanisms of PARPi resistance. Compared with BRCA1-proficient cells, PARPi-resistant BRCA1-deficient cells are increasingly dependent on ATR for survival. ATR inhibitors (ATRis) disrupt BRCA1-independent RAD51 loading to DSBs and stalled forks in PARPi-resistant BRCA1-deficient cells, overcoming both resistance mechanisms. In tumor cells derived from patients, ATRis also overcome the bypass of BRCA1/2 in fork protection. Thus, ATR inhibition is a unique strategy to overcome the PARPi resistance of BRCA-deficient cancers