The 9-1-1 checkpoint clamp stimulates DNA resection by Dna2-Sgs1 and Exo1

Abstract

Single-stranded DNA (ssDNA) at DNA ends is an important regulator of the DNA damage response. Resection, the generation of ssDNA, affects DNA damage checkpoint activation, DNA repair pathway choice, ssDNA-associated mutation and replication fork stability. In eukaryotes, extensive DNA resection requires the nuclease Exo1 and nuclease/helicase pair: Dna2 and Sgs1(BLM). How Exo1 and Dna2-Sgs1(BLM) coordinate during resection remains poorly understood. The DNA damage checkpoint clamp (the 9-1-1 complex) has been reported to play an important role in stimulating resection but the exact mechanism remains unclear. Here we show that the human 9-1-1 complex enhances the cleavage of DNA by both DNA2 and EXO1 in vitro, showing that the resection-stimulatory role of the 9-1-1 complex is direct. We also show that in Saccharomyces cerevisiae, the 9-1-1 complex promotes both Dna2-Sgs1 and Exo1-dependent resection in response to uncapped telomeres. Our results suggest that the 9-1-1 complex facilitates resection by recruiting both Dna2-Sgs1 and Exo1 to sites of resection. This activity of the 9-1-1 complex in supporting resection is strongly inhibited by the checkpoint adaptor Rad9(53BP1). Our results provide important mechanistic insights into how DNA resection is regulated by checkpoint proteins and have implications for genome stability in eukaryotes.

Figure 1.

Human 9-1-1 complex stimulates DNA2 and EXO1 activities. (A, B) DNA2 cleavage activities on a fork (A) and a flap (B) substrate in the presence of the 9-1-1 complex or PCNA. Labeling of the substrate at the 3′ end allows the determination of the final products of the reaction. Products are analyzed on alkaline gels as described in the Materials and Methods section. (C, D) EXO1 cleavage activities on a 3′overhang (C) and a flap (D) structure in the presence of 9-1-1 or PCNA. Substrates used for each panel of the experiment are depicted on the top of the gel with the asterisk indicating the site of the ³²P label. The flap is 30 nt long. The labeled strand in panel (C) is 60 nt long.

Figure 2.

Human 9-1-1 complex loads onto DNA to stimulate nuclease activity. (A) DNA2 cleavage activity on a 5′ flap substrate in the presence of 9-1-1 and RAD17/RFC. (B) Binding efficiency of 9-1-1 (low and high concentration) in the presence of the clamp loader RAD17/RFC on a 5′ flap substrate. (C) DNA2 nuclease activity in the presence of the 9-1-1 complex on a substrate containing blocked template ends and free 5′ flap. Substrates used for each panel of experiment are depicted on the top of the gel with the asterisk indicating the site of the ³²P label.

Figure 3.

The 9-1-1 complex promotes extensive DNA resection. (A) Maps of the right arms of chromosomes VI. (B) Analyses of 3′ssDNA accumulation following telomere uncapping in strains lacking Mec3 and/or Rad9 (all in cdc13-1 cdc15-2 bar1 background). The data and error bars plotted are means and standard deviations from two independent experiments.

Figure 4.

The 9-1-1 complex promotes Exo1-dependent resection. (A–C) Analyses of 3′ssDNA accumulation following telomere uncapping in strains lacking Mec3, Sgs1 and/or Exo1 (all in cdc13-1 cdc15-2 bar1 background).The data and error bars are the means and standard deviations from individual samples measured in triplicate, except RAD+ and mec3Δ strains, where the data and error bars plotted are means and standard deviations from two independent strains.

Figure 5.

The 9-1-1 complex promotes Exo1- and Dna2-Sgs1-dependent resection. (A) Analyses of 3′ssDNA accumulation following telomere uncapping in rad9Δ strains lacking Mec3, Sgs1 and/or Exo1 (all in cdc13-1 cdc15-2 bar1 background). The data and error bars plotted are means and standard deviations from two independent experiments. (B) Analyses of 3′ssDNA accumulation following telomere uncapping in strains lacking Mec3, Rad9 and or Dna2 (all in cdc13-1 cdc15-2 bar1 background). The data and error bars are the means and standard deviations from individual samples measured in triplicate.

Figure 6.

The 9-1-1 complex promotes the binding of Dna2, Sgs1 and Exo1 to DNA following telomere uncapping. (A) Co-immunoprecipitation experiment to detect interaction between 9-1-1 and Dna2/Sgs1/Exo1. Protein extract from cells expressing (+) or not expressing (−) the indicated epitope-tagged proteins was subjected to immunoprecipitation with anti-HA (left panel) or anti-Myc (right panel) antibodies, before probing with anti-HA (top panels) or anti-Myc (bottom panels) antibodies. (B) Two-hybrid analysis in cdc13-1 reporter strains at 23°C and 27°C. Bait plasmid contains either RAD17 or MEC3 and prey plasmids contain the sequence of the genes indicated. (C–E) ChIP analyses of Dna2-Myc, Sgs1-Myc and Exo1-Myc binding to DUG1 and PDA1 following telomere uncapping. The data plotted are fold increase over a control locus PAC2 and represent the means and standard deviations (error bars) from individual samples measured in triplicate.

Figure 7.

The role of the 9-1-1 complex in stimulating DNA resection. (A, B) The 9-1-1 complex stimulates extensive DNA resection by recruiting Dna2-Sgs1^BLM and Exo1 to sites of resection. Following recruitment by 9-1-1, Dna2-Sgs1^BLM and Exo1 contribute differently to resection. In the presence of Rad9^53BP1(A), extensive resection is more dependent on Exo1 than Dna2-Sgs1^BLM. In the absence of Rad9^53BP1(B), extensive resection is more dependent on Dna2-Sgs1^BLM than Exo1. The set of four filled ellipses represent histone octamers.