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The Fanconi Anemia Pathway Maintains Genome Stability by Coordinating Replication and Transcription.

Schwab RA, Nieminuszczy J, Shah F, Langton J, Lopez Martinez D, Liang CC, Cohn MA, Gibbons RJ, Deans AJ, Niedzwiedz W - Mol. Cell (2015)

Bottom Line: However, how these proteins limit replication stress remains largely elusive.Here we show that conflicts between replication and transcription activate the FA pathway.Finally, we demonstrate that the molecular mechanism by which the FA pathway limits R-loop accumulation requires FANCM translocase activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncology, Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DS, UK.

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Related in: MedlinePlus

The FA Pathway Prevents DNA:RNA Hybrid Accumulation via the DNA:RNA Branch Migration Activity of FANCM(A) Quantified immunofluorescence intensity with the DNA:RNA hybrid-specific antibody S9.6 of sictrl or siFANCM-treated cells. The dot plot represents the mean fluorescence intensity of individual nuclei from three experiments, with the middle line representing the mean and whiskers the SEM (two-tailed Mann-Whitney test). ∗∗∗∗p ≤ 0.0001.(B) Model of branch migration activity of FANCM, leading to the resolution of DNA:RNA hybrids, and in vitro unwinding assays with purified FANCM or FANCM K117R mutant and FAAP24 on migratable 3′ and 5′ DNA:RNA flap structures.(C) Quantification of DNA:RNA hybrid resolution using a migratable replication (Rep) fork substrate as a positive control.(D) Quantified immunofluorescence intensity with the DNA:RNA hybrid-specific antibody S9.6 of WT DT40, FANCM−/− cells, and DT40 cells expressing FANCM D203A translocase-dead mutant protein. The dot plot represents mean fluorescence intensity of individual nuclei from three independent experiments, with the middle line representing the mean and whiskers the SEM (two-tailed Mann-Whitney test). ∗∗∗∗p ≤ 0.0001.(E) Model explaining how the FA pathway prevents conflicts between replication and transcription. In the absence of the FA pathway, conflicts between replication and transcription result in activation of the DDR, DNA:RNA hybrid accumulation, defects in replication fork progression, DNA lesions, and genomic instability. In the presence of a functional FA pathway, transcription-induced replication fork stalling leads to monoubiquitination of FANCD2 by the FA core complex proteins and, therefore, activation of the FA pathway, resulting in stabilization of stalled replication forks. Subsequently, FANCM resolves replication blocks consisting of DNA:RNA hybrids via its translocase activity, and replication can resume normally.
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fig6: The FA Pathway Prevents DNA:RNA Hybrid Accumulation via the DNA:RNA Branch Migration Activity of FANCM(A) Quantified immunofluorescence intensity with the DNA:RNA hybrid-specific antibody S9.6 of sictrl or siFANCM-treated cells. The dot plot represents the mean fluorescence intensity of individual nuclei from three experiments, with the middle line representing the mean and whiskers the SEM (two-tailed Mann-Whitney test). ∗∗∗∗p ≤ 0.0001.(B) Model of branch migration activity of FANCM, leading to the resolution of DNA:RNA hybrids, and in vitro unwinding assays with purified FANCM or FANCM K117R mutant and FAAP24 on migratable 3′ and 5′ DNA:RNA flap structures.(C) Quantification of DNA:RNA hybrid resolution using a migratable replication (Rep) fork substrate as a positive control.(D) Quantified immunofluorescence intensity with the DNA:RNA hybrid-specific antibody S9.6 of WT DT40, FANCM−/− cells, and DT40 cells expressing FANCM D203A translocase-dead mutant protein. The dot plot represents mean fluorescence intensity of individual nuclei from three independent experiments, with the middle line representing the mean and whiskers the SEM (two-tailed Mann-Whitney test). ∗∗∗∗p ≤ 0.0001.(E) Model explaining how the FA pathway prevents conflicts between replication and transcription. In the absence of the FA pathway, conflicts between replication and transcription result in activation of the DDR, DNA:RNA hybrid accumulation, defects in replication fork progression, DNA lesions, and genomic instability. In the presence of a functional FA pathway, transcription-induced replication fork stalling leads to monoubiquitination of FANCD2 by the FA core complex proteins and, therefore, activation of the FA pathway, resulting in stabilization of stalled replication forks. Subsequently, FANCM resolves replication blocks consisting of DNA:RNA hybrids via its translocase activity, and replication can resume normally.

Mentions: Next, we asked whether the FA pathway could provide enzymatic activity to resolve DNA:RNA hybrids directly. A likely member of the FA pathway with such a putative function is FANCM. It possesses double-stranded DNA translocase activity implicated in the processing of Holliday junction intermediates and replication fork reversal in vitro (Gari et al., 2008). In vivo, the protein has been shown to rescue stalled forks (Blackford et al., 2012, Schwab et al., 2010). Studies using recombinant FANCM have tested its activity only with DNA:DNA substrates (Gari et al., 2008). However, the protein is, in fact, classified to belong to the DEAD/DEAH family of DNA:RNA helicases. Therefore, we considered the possibility that FANCM could directly remove DNA:RNA hybrids through its translocase activity. In line with this notion, we observed a significant increase in DNA:RNA hybrid formation in FANCM-depleted cells (Figure 6A; Figure S6A). Importantly, purified FANCM was not only able to unwind replication fork structures, as shown previously (Gari et al., 2008; Figure S6B), but it efficiently unwound DNA:RNA hybrids in vitro (Figures 6B and 6C) despite such substrates being more stable than DNA:DNA hybrids found at a replication fork (Chien and Davidson, 1978). The branch-migratable structures were designed to mimic both the 5′ and 3′ ends of a DNA:RNA hybrid, and our biochemical analyses have shown that FANCM can translocate along either the Watson or Crick strand in a 3′-5′ direction and disrupt DNA:RNA base pairing (Figures 6B and 6C). As expected, the resolution of DNA:RNA hybrids requires FANCM’s translocase activity because the translocase-dead mutant protein was unable to unwind these substrates. Similarly, addition of non-hydrolysable ATP (ATP-γ-S) blocked the reaction (Figures 6B and 6C; Figure S6B). Finally, knockin DT40 cells expressing the translocase-dead variant of FANCM (Rosado et al., 2009) also displayed elevated levels of DNA:RNA hybrids (Figure 6D). This suggests a mechanism by which FANCM directly promotes DNA:RNA hybrid resolution, replication fork restart, and, consequently, faithful genome duplication. Because we observed no unwinding activity when the RNA sequence and flap sequence were heterologous (Figure S6C), we conclude that DNA:RNA hybrid resolution is carried out via its branch migration activity.


The Fanconi Anemia Pathway Maintains Genome Stability by Coordinating Replication and Transcription.

Schwab RA, Nieminuszczy J, Shah F, Langton J, Lopez Martinez D, Liang CC, Cohn MA, Gibbons RJ, Deans AJ, Niedzwiedz W - Mol. Cell (2015)

The FA Pathway Prevents DNA:RNA Hybrid Accumulation via the DNA:RNA Branch Migration Activity of FANCM(A) Quantified immunofluorescence intensity with the DNA:RNA hybrid-specific antibody S9.6 of sictrl or siFANCM-treated cells. The dot plot represents the mean fluorescence intensity of individual nuclei from three experiments, with the middle line representing the mean and whiskers the SEM (two-tailed Mann-Whitney test). ∗∗∗∗p ≤ 0.0001.(B) Model of branch migration activity of FANCM, leading to the resolution of DNA:RNA hybrids, and in vitro unwinding assays with purified FANCM or FANCM K117R mutant and FAAP24 on migratable 3′ and 5′ DNA:RNA flap structures.(C) Quantification of DNA:RNA hybrid resolution using a migratable replication (Rep) fork substrate as a positive control.(D) Quantified immunofluorescence intensity with the DNA:RNA hybrid-specific antibody S9.6 of WT DT40, FANCM−/− cells, and DT40 cells expressing FANCM D203A translocase-dead mutant protein. The dot plot represents mean fluorescence intensity of individual nuclei from three independent experiments, with the middle line representing the mean and whiskers the SEM (two-tailed Mann-Whitney test). ∗∗∗∗p ≤ 0.0001.(E) Model explaining how the FA pathway prevents conflicts between replication and transcription. In the absence of the FA pathway, conflicts between replication and transcription result in activation of the DDR, DNA:RNA hybrid accumulation, defects in replication fork progression, DNA lesions, and genomic instability. In the presence of a functional FA pathway, transcription-induced replication fork stalling leads to monoubiquitination of FANCD2 by the FA core complex proteins and, therefore, activation of the FA pathway, resulting in stabilization of stalled replication forks. Subsequently, FANCM resolves replication blocks consisting of DNA:RNA hybrids via its translocase activity, and replication can resume normally.
© Copyright Policy - CC BY
Related In: Results  -  Collection

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Show All Figures
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fig6: The FA Pathway Prevents DNA:RNA Hybrid Accumulation via the DNA:RNA Branch Migration Activity of FANCM(A) Quantified immunofluorescence intensity with the DNA:RNA hybrid-specific antibody S9.6 of sictrl or siFANCM-treated cells. The dot plot represents the mean fluorescence intensity of individual nuclei from three experiments, with the middle line representing the mean and whiskers the SEM (two-tailed Mann-Whitney test). ∗∗∗∗p ≤ 0.0001.(B) Model of branch migration activity of FANCM, leading to the resolution of DNA:RNA hybrids, and in vitro unwinding assays with purified FANCM or FANCM K117R mutant and FAAP24 on migratable 3′ and 5′ DNA:RNA flap structures.(C) Quantification of DNA:RNA hybrid resolution using a migratable replication (Rep) fork substrate as a positive control.(D) Quantified immunofluorescence intensity with the DNA:RNA hybrid-specific antibody S9.6 of WT DT40, FANCM−/− cells, and DT40 cells expressing FANCM D203A translocase-dead mutant protein. The dot plot represents mean fluorescence intensity of individual nuclei from three independent experiments, with the middle line representing the mean and whiskers the SEM (two-tailed Mann-Whitney test). ∗∗∗∗p ≤ 0.0001.(E) Model explaining how the FA pathway prevents conflicts between replication and transcription. In the absence of the FA pathway, conflicts between replication and transcription result in activation of the DDR, DNA:RNA hybrid accumulation, defects in replication fork progression, DNA lesions, and genomic instability. In the presence of a functional FA pathway, transcription-induced replication fork stalling leads to monoubiquitination of FANCD2 by the FA core complex proteins and, therefore, activation of the FA pathway, resulting in stabilization of stalled replication forks. Subsequently, FANCM resolves replication blocks consisting of DNA:RNA hybrids via its translocase activity, and replication can resume normally.
Mentions: Next, we asked whether the FA pathway could provide enzymatic activity to resolve DNA:RNA hybrids directly. A likely member of the FA pathway with such a putative function is FANCM. It possesses double-stranded DNA translocase activity implicated in the processing of Holliday junction intermediates and replication fork reversal in vitro (Gari et al., 2008). In vivo, the protein has been shown to rescue stalled forks (Blackford et al., 2012, Schwab et al., 2010). Studies using recombinant FANCM have tested its activity only with DNA:DNA substrates (Gari et al., 2008). However, the protein is, in fact, classified to belong to the DEAD/DEAH family of DNA:RNA helicases. Therefore, we considered the possibility that FANCM could directly remove DNA:RNA hybrids through its translocase activity. In line with this notion, we observed a significant increase in DNA:RNA hybrid formation in FANCM-depleted cells (Figure 6A; Figure S6A). Importantly, purified FANCM was not only able to unwind replication fork structures, as shown previously (Gari et al., 2008; Figure S6B), but it efficiently unwound DNA:RNA hybrids in vitro (Figures 6B and 6C) despite such substrates being more stable than DNA:DNA hybrids found at a replication fork (Chien and Davidson, 1978). The branch-migratable structures were designed to mimic both the 5′ and 3′ ends of a DNA:RNA hybrid, and our biochemical analyses have shown that FANCM can translocate along either the Watson or Crick strand in a 3′-5′ direction and disrupt DNA:RNA base pairing (Figures 6B and 6C). As expected, the resolution of DNA:RNA hybrids requires FANCM’s translocase activity because the translocase-dead mutant protein was unable to unwind these substrates. Similarly, addition of non-hydrolysable ATP (ATP-γ-S) blocked the reaction (Figures 6B and 6C; Figure S6B). Finally, knockin DT40 cells expressing the translocase-dead variant of FANCM (Rosado et al., 2009) also displayed elevated levels of DNA:RNA hybrids (Figure 6D). This suggests a mechanism by which FANCM directly promotes DNA:RNA hybrid resolution, replication fork restart, and, consequently, faithful genome duplication. Because we observed no unwinding activity when the RNA sequence and flap sequence were heterologous (Figure S6C), we conclude that DNA:RNA hybrid resolution is carried out via its branch migration activity.

Bottom Line: However, how these proteins limit replication stress remains largely elusive.Here we show that conflicts between replication and transcription activate the FA pathway.Finally, we demonstrate that the molecular mechanism by which the FA pathway limits R-loop accumulation requires FANCM translocase activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncology, Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DS, UK.

Show MeSH
Related in: MedlinePlus