The Fanconi Anemia Pathway Maintains Genome Stability by Coordinating Replication and Transcription.
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.
Affiliation: Department of Oncology, Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DS, UK.Show MeSH
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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.
Affiliation: Department of Oncology, Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DS, UK.