DNA2 and MSH2 activity collectively mediate chemically stabilized G4 for efficient telomere replication

Abstract

G-quadruplexes (G4s) are widely existing stable DNA secondary structures in mammalian cells. A long-standing hypothesis is that timely resolution of G4s is needed for efficient and faithful DNA replication. In vitro, G4s may be unwound by helicases or alternatively resolved via DNA2 nuclease mediated G4 cleavage. However, little is known about the biological significance and regulatory mechanism of the DNA2-mediated G4 removal pathway. Here, we report that DNA2 deficiency or its chemical inhibition leads to a significant accumulation of G4s and stalled replication forks at telomeres, which is demonstrated by a high-resolution technology: Single molecular analysis of replicating DNA (SMARD). We further identify that the DNA repair complex MutSα (MSH2-MSH6) binds G4s and stimulates G4 resolution via DNA2-mediated G4 excision. MSH2 deficiency, like DNA2 deficiency or inhibition, causes G4 accumulation and defective telomere replication. Meanwhile, G4-stabilizing environmental compounds block G4 unwinding by helicases but not G4 cleavage by DNA2. Consequently, G4 stabilizers impair telomere replication and cause telomere instabilities, especially in cells deficient in DNA2 or MSH2.

Fig. 1 |. DNA2 deficiency leads to G4 accumulation in mammalian cells

a AIRYSCAN confocal microscopy of G4s; DNA2-G4 co-immunofluorescence staining in MEFs. The right panel shows the enlarged view of the boxed region. The spots with co-localized DNA2 and G4 foci were indicated with white arrows. Scale bar = 5 μm; b Reconstituted G4 excision repair assay. Top panel: schematic diagram elucidating biochemical assays for G4 excision, in which nucleases DNA2 cleave G4 to convert it into a gap. DNA polymerase such as Polβ, Polδ, or Polε then fills the gap using ³²P-dTTP and the other three dNTP as substrates, producing ³²P-labeled products. Bottom panel: Representative denaturing PAGE image showing G4 excision by DNA2 and the gap filling by Polβ; c, d G4 immunofluorescence staining in WT and DNA2^+/− MEFs. Panel c shows the representative AIRYSCAN confocal microscopy images of G4s, and Panel (d) shows the quantification of G4 in different cells. All p values were calculated using the Student’s t-test. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. Scale bar = 5 μm; e-g DNA replication at telomeres in WT and DNA2^+/− MEFs; (e) Top: Scheme of the IdU (green) and CldU (red) pulse labeling. Bottom: SMARD microscopy images of replicated telomeres in WT and DNA2^+/− MEF cells. Imaged fibers contain both non-telomeric and telomeric DNA. SMARD fibers are arranged showing non-telomeric DNA at the left and telomeres on the right (indicated by the blue telomere drawing) aligned by start of the telomere at the yellow line. The top panels of SMARD fibers represent fully replicated telomeres with either IdU and/or CldU incorporation along the length of the telomere. Middle panels depict fibers with partial telomere replication, where IdU or CldU incorporated into some of the telomere but not for the entire length. The bottom panels depict completely un-replicated telomeres, where IdU and CldU incorporated immediately adjacent to, but not into telomeres. (f) Relative length of replicated telomeres in different cells. (g) Percentage of incompletely replicated telomeres in different cells. The frequency of partial telomere replication and replication fork stalling adjacent to telomeres were calculated.

Fig. 2 |. G4 binding protein complex MutSα stimulates DNA2-mediated G4 cleavage.

a Silver staining SDS-PAGE showing the pulled-down proteins by Flag-tag M2 beads in whole cell extracts from 293T cells transfect with or empty vector of the vector encoding 3x-Flag human DNA2; b DNA replication and repair proteins co-pulled down with 3x-Flag-tagged DNA2; c, d Co-IP and western blot analysis of DNA2 interaction with MSH2 and MSH6; e The EMSA image shows the MutSα rather than XPE complex binds to the ³²P-label G4 substrate. It also shows that DNA2 cleaves the G4 substrate; f AIRYSCAN confocal microscopy of G4s; MSH2-G4 co-immunofluorescence staining in MEFs. The right panel shows the enlarged view of the boxed region. The spots with co-localized MSH2 and G4 foci were indicated with white arrows. Scale bar = 5 μm; g The representative denaturing PAGE image shows DNA2 cleaving the ³²P-labeled G4 substrate in the absence or presence of MutSα; h The representative denaturing PAGE image shows cleavage of the ³²P-labeled G4 substrate by the DNA2-MutSα complex in the absence or presence of G4 stabilizer TMPyP4.

Fig. 3 |. MSH2 deficiency results in G4 accumulation, defects in telomere replication.

a, b G4 immunofluorescence staining in WT and MSH2^−/− MEFs. Panel A shows the representative AIRYSCAN confocal microscopy images of G4s, and Panel B shows the quantification of G4 in WT and MSH2^−/− MEFs. Scale bar = 5 μm; c-e SMARD analysis of WT and MSH2^−/− MEF cells. (c) Representative microscopy images of the replicated telomeres in WT or MSH2^−/−; (d) Quantification of the relative length of replicated telomeres in different cells; and (e) Percentage of partial telomere replication and replication fork stalling adjacent to telomeres in WT or MSH2^−/− cells; f Telomere FISH images showing telomere abnormalities in the WT and MSH2^−/− MEFs. Scale bar = 5 μm; g Quantification of fragile telomeres in WT or MSH2^−/− cells treated with PIPER and capreomycin. All p values were calculated using the Two-way ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Fig. 4 |. G4-stabilizing ECCs inhibit FANCJ unwinding G4, but not DNA2-mediated cleavage of G4 in vitro.

a, b Primer extension on the non-G4 or G4 template in the presence of increasing concentrations of G4-stabilizing ECCs PIPER (a) and capreomycin (b). The top shows the diagram of the Polδ-catalyzed primer extension on a DNA template without or with a G4-forming sequence. The primer was labeled with FAM on the 5’ end. The bottom shows the representative denaturing-PAGE image of the primer extension assay; c, d Primer extension on the G4 template in the absence or presence of 5 nM FANCJ with or without 10, 50 and 100 μM PIPER (c) or capreomycin (d); e, f The cleavage of a FAM-labeled G4 substrate with increasing concentration of DNA2 in the absence or presence of increasing concentrations of PIPER (e) or capreomycin (f); g, h G4 immunofluorescence staining in WT and MSH2^−/− MEFs. (g) shows the representative AIRYSCAN confocal microscopy images of G4s, and (h) shows the quantification of G4 in WT, DNA2^+/−, or MSH2^−/− MEFs without or with treatment with G4 stabilizers PIPER or capreomycin. Scale bar = 5 μm.

Fig. 5 |. G4-stabilizing ECCs impair DNA replication at telomeres in WT DNA2^+/− and MSH2^−/− MEF cells.

a SMARD microscopy images of replicated telomeres in WT, DNA2^+/−, and MSH2^−/− MEF cells with or without exposure to PIPER or capreomycin; b Relative length of replicated telomeres in different cells; c Percentage of incompletely replicated telomeres in different cells. The frequency of partial telomere replication and replication fork stalling adjacent to telomeres was calculated.

Fig. 6 |. G4-stabilizing ECCs enhance telomere abnormalities in WT DNA2^+/− and MSH2^−/− MEF cells.

a Telomere FISH images showing telomere abnormalities in the WT and DNA2^+/− MEFs treated with PIPER and capreomycin. Scale bar = 5 μm; b Quantification of fragile telomeres and shortened telomeres in different cells; c Telomere FISH images showing telomere abnormalities in the WT and MSH2^−/− MEFs treated with PIPER and capreomycin. Scale bar = 5 μm; d Quantification of fragile telomeres and shortened telomeres in different cells. All p values were calculated using the Two-way ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.