Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2014 Sep 26;289(39):27314-26.
doi: 10.1074/jbc.M114.578823. Epub 2014 Aug 13.

DNA2 Cooperates With the WRN and BLM RecQ Helicases to Mediate Long-Range DNA End Resection in Human Cells

Affiliations
Free PMC article

DNA2 Cooperates With the WRN and BLM RecQ Helicases to Mediate Long-Range DNA End Resection in Human Cells

Andreas Sturzenegger et al. J Biol Chem. .
Free PMC article

Abstract

The 5'-3' resection of DNA ends is a prerequisite for the repair of DNA double strand breaks by homologous recombination, microhomology-mediated end joining, and single strand annealing. Recent studies in yeast have shown that, following initial DNA end processing by the Mre11-Rad50-Xrs2 complex and Sae2, the extension of resection tracts is mediated either by exonuclease 1 or by combined activities of the RecQ family DNA helicase Sgs1 and the helicase/endonuclease Dna2. Although human DNA2 has been shown to cooperate with the BLM helicase to catalyze the resection of DNA ends, it remains a matter of debate whether another human RecQ helicase, WRN, can substitute for BLM in DNA2-catalyzed resection. Here we present evidence that WRN and BLM act epistatically with DNA2 to promote the long-range resection of double strand break ends in human cells. Our biochemical experiments show that WRN and DNA2 interact physically and coordinate their enzymatic activities to mediate 5'-3' DNA end resection in a reaction dependent on RPA. In addition, we present in vitro and in vivo data suggesting that BLM promotes DNA end resection as part of the BLM-TOPOIIIα-RMI1-RMI2 complex. Our study provides new mechanistic insights into the process of DNA end resection in mammalian cells.

Keywords: DNA Damage; DNA Helicase; DNA Recombination; DNA Repair; Genomic Instability; RecQ.

Figures

FIGURE 1.
FIGURE 1.
DNA end resection by DNA2 and WRN. A, SDS-PAGE analysis of purified WRN (0.7 μg) and DNA2 (0.4 μg) proteins. Gel was stained with Coomassie Brilliant Blue R-250. The molecular weights of protein standards are indicated on the left. B, schematic of the DNA end resection assay. The resection products were either left untreated or hybridized with synthetic 32P-labeled oligonucleotides complementary to the 3′-terminated strand. DNA species were separated by electrophoresis in a 1% agarose gel and visualized by SYBR gold staining and phosphorimaging, respectively. Probes complementary to the regions spanning nt positions 112–133 and 353–374 (relative to the 3′ end) were used in this study. Only a part of the DNA substrate is shown. C, time course of resection of 3′-tailed (26 nt) and blunt-ended DNA substrates by DNA2 and WRN. Reactions were carried out at 37 °C and contained 2 nm DNA, 350 nm RPA, 10 nm WRN, and 8 nm DNA2. Reaction products at the indicated time points were analyzed as outlined in B. The 112- to 133-nt probe was used in this analysis. Lane 1, heat-denatured substrate; lane 14, 3′-tailed substrate incubated with 20 nm EXO1 and 350 nm RPA for 2 min (R1); lane 15, blunt-ended substrate incubated with 20 nm EXO1 and 350 nm RPA for 2 min (R2). D, directionality of DNA end resection by WRN-DNA2. Reactions were carried out at 37 °C for 60 min and contained 2 nm 3′-tailed DNA substrate, 350 nm RPA, 8 nm DNA2, and 10 nm WRN. Resection products were annealed with radiolabeled oligonucleotide probes complementary to either 3′-terminated (position 353–374 nt relative to the 3′ end) or 5′-terminated (position 353–374 nt relative to 3′ end) strand and analyzed as in C. E, 5′ end resection of 3′-tailed DNA substrate by WRN-DNA2 is dependent on WRN concentration and the presence of ATP and RPA. Reactions were carried out at 37 °C for 60 min and contained, as indicated, 2 nm DNA, 350 nm RPA, 1 mm ATP, 8 nm DNA2, and different WRN concentrations. Resection products were detected using the 112–133-nt probe. F, dependence of WRN-DNA2-catalyzed resection of 3′-tailed substrate on WRN concentration. Resection at the positions of 112–133 nt and 353–374 nt from the 3′ end of the DNA substrate was monitored. Reactions were carried out as in E. Relative concentration of the resection product generated by WRN-DNA2 at each WRN concentration was calculated as a percentage of the product generated by 20 nm EXO1 after 2 min. Data are mean ± S.D. (n = 3).
FIGURE 2.
FIGURE 2.
5′ end resection of 3′-tailed DNA substrate by WRN-DNA2 depends on the helicase activity of WRN and the nuclease activity of DNA2. Reactions were carried out at 37 °C for 60 min and contained 2 nm DNA, 350 nm RPA, 1 mm ATP, 8 nm DNA2, and 10 nm WRN. Resection products were detected using the 112–133 nt probe. WRNHD, helicase-deficient mutant of WRN (K567M); WRNND, nuclease-deficient mutant of WRN (E84A); DNA2HD, helicase-deficient mutant of DNA2 (K654R); DNA2ND, nuclease-deficient mutant of DNA2 (D277A).
FIGURE 3.
FIGURE 3.
Physical interaction between DNA2 and WRN in vitro and in vivo. A, coimmunoprecipitation of WRN with DNA2 from human cells. HEK293 cells were transfected with vectors expressing FLAG-DNA2 and WRN as indicated. Cell extracts were immunoprecipitated (IP) with anti-FLAG antibody as described under “Experimental Procedures.” Blots were probed with the indicated antibodies. 5% of input material was loaded. B, effect of DNA damage on the formation of DNA2-WRN and DNA2-BLM complexes in human cells. HEK293 cells stably transfected with the FLAG-DNA2 construct (HEK293-D) were treated with 1 μm CPT. At the indicated time points, complex formation between FLAG-DNA2 and endogenous WRN and BLM, respectively, was tested by immunoprecipitation using anti-FLAG antibody. C, coimmunoprecipitation of DNA2 with WRN from human cells. Extracts from HEK293-D cells were subjected to immunoprecipitation with anti-WRN antibody or control IgG. The immunoprecipitates were tested for the presence of FLAG-DNA2 and WRN by Western blotting. As a control, a WRN immunoprecipitate from HEK293 cells was also analyzed (lane 3). D, coimmunoprecipitation of DNA2 with WRN from a mixture of purified proteins. DNA2 (500 ng) was incubated with or without WRN (500 ng) at 4 °C for 4 h. The mixtures were subjected to immunoprecipitation with anti-WRN antibody. E, domain organization of WRN. Exo, exonuclease domain; Zn, zinc-binding domain; WH, winged-helix domain; HRDC, helicase and RNaseD C-terminal domain. Black lines indicate WRN fragments used for mapping the DNA2-interaction site on WRN. F, GST pulldown assay. Glutathione beads coated with the indicated GST-tagged fragments of WRN were incubated with purified His6-DNA2-FLAG protein at 4 °C for 2 h, and bound proteins were analyzed by Western blotting as described under “Experimental Procedures.” 1% of input was loaded in B and C, whereas 10% of input was loaded in D and F.
FIGURE 4.
FIGURE 4.
Comparison of DNA end resection activities of WRN-DNA2 and BLM-DNA2. A, comparison of helicase activities of WRN and BLM. Reactions contained 1 nm 32P-labeled forked DNA duplex (inset) and different concentrations of WRN or BLM. Reactions were incubated at 37 °C for 30 min, and reaction products were quantified as described under “Experimental Procedures.” Data are mean ± S.D. (n = 3). B, time course of resection of 3′-tailed DNA substrate catalyzed by WRN-DNA2 and BLM-DNA2, respectively. Reactions contained 2 nm DNA, 350 nm RPA, 8 nm DNA2, and 10 nm WRN/BLM. Reaction aliquots withdrawn at the indicated time points were subjected to electrophoresis on a 1% agarose gel after hybridization of radiolabeled probes complementary to 3′-terminated strand at the indicated positions. Radiolabeled DNA species were visualized by phosphorimaging. C, quantification of the reactions in B. Relative concentration of resection products generated at each time point was calculated as a percentage of the product generated by 20 nm EXO1 after 2 min. Data are mean ± S.D. (n = 3). D, processing of 3′-tailed (26 nt) and blunt-ended DNA substrates in reactions with indicated composition. Reactions were carried out at 37 °C for 60 min and contained 2 nm DNA, 350 nm RPA, and, where indicated, 8 nm DNA2, 20 nm WRN, and 20 nm BLM. Reaction products were analyzed as in Fig. 1C. Lane 1, heat-denatured substrate; lane 14, 3′-tailed substrate incubated with 20 nm EXO1 for 2 min (R1); lane 15, blunt-ended substrate incubated with 20 nm EXO1 for 2 min (R2).
FIGURE 5.
FIGURE 5.
WRN and BLM interact epistatically with DNA2 to promote DSB repair by SSA in human cells. A, schematic of the SA-GFP reporter cassette. SSA-mediated repair of a DSB at the I-SceI-cutting site results in the formation of a functional GFP allele. B, efficiency of SSA-mediated repair of I-SceI-induced DSB in HEK293/SA-GFP cells treated with the indicated siRNAs. Cells were transfected with the appropriate siRNAs (40 nm) 2 days prior to transfection of the I-SceI-expressing plasmid. The percentage of GFP-positive cells in each sample was measured by flow cytometry 2 days after I-SceI plasmid transfection and taken as a measure of DSB repair efficiency. The plotted values represent the relative repair efficiency calculated as a percentage of repair efficiency measured in cells transfected with control siRNA (siLuc, 100%). Data are mean ± S.D. (n ≥ 3). C, Western blot analysis of extracts from HEK293/SA-GFP cells transfected with indicated siRNAs under the same conditions as for SA-GFP reporter assays. Blots were probed with the indicated antibodies. D, rescue of the SSA-repair defect of WRN-depleted HEK293/SA-GFP cells by expression of the siRNA-resistant variant of WRN. An SA-GFP reporter assay was performed as in B. The WRN plasmid (WRN) or empty vector (EV) were cotransfected with the I-SceI plasmid. E, efficiency of SSA-mediated repair of I-SceI-induced DSB in U2OS/SA-GFP cells treated with the indicated siRNAs. Experiments were performed as in B. F, Western blot analysis of extracts from U2OS/SA-GFP cells transfected with the indicated siRNAs. Blots were probed with the indicated antibodies. G, quantitative real-time PCR showing that EXO1 mRNA levels are down-regulated by specific siRNA. Data are mean ± S.D. (n = 3).
FIGURE 6.
FIGURE 6.
DNA2, WRN, and BLM act in the same pathway of DSB end resection. A, frequency of camptothecin-induced RPA foci in nuclei of U2OS cells depleted of the indicated proteins. Cells were transfected with appropriate siRNAs and, 48 h later, treated with 1 μm camptothecin for 1 h. Cells were then detergent-extracted and fixed with formaldehyde. RPA and γ-H2AX (a marker of DNA damage) were visualized by indirect immunofluorescence. DAPI was used to stain nuclei. The average number of RPA foci per γ-H2AX-positive cell was determined for each sample using an Olympus Scan̂R screening station. The data points are mean ± S.D. (n = 3). B, Western blot analysis of extracts from U2OS cells transfected with indicated siRNAs. Blots were probed with the antibodies indicated on the right.
FIGURE 7.
FIGURE 7.
Involvement of TOPOIIIα, RMI1, and RMI2 in DNA end resection. A, SDS-PAGE analysis of purified TRR complex (1.5 μg) and BLM (0.5 μg). The gel was stained with Coomassie Brilliant Blue R-250. The molecular weights of protein standards are indicated on the left. B, stimulation of BLM-DNA2-catalyzed DNA end resection by the TRR complex. Reactions contained 2 nm 3′-tailed pUC19 substrate, 8 nm DNA2, 10 nm BLM, 350 nm RPA, and varying concentrations of TRR. BLM and TRR were preincubated for 5 min on ice prior to addition to the reaction. Reaction products were analyzed as in Fig. 1C using the 112–133-nt probe. C, quantification of the product of reactions in B. Data are mean ± S.D. (n = 3). The data are normalized to the amount of product in the reaction containing only BLM and DNA2 (100%). D, RMI1 acts epistatically with BLM and DNA2 to promote DSB repair by SSA in human cells. Efficiency of SSA-mediated repair of I-SceI-induced DSB in U2OS/SA-GFP cells transfected with indicated siRNAs was measured as in Fig. 5E. E, Western blot analysis of extracts from U2OS/SA-GFP cells transfected with indicated siRNAs under the same conditions as for SA-GFP reporter assays. Blots were probed with the indicated antibodies.

Similar articles

See all similar articles

Cited by 64 articles

See all "Cited by" articles

Publication types

MeSH terms

Feedback