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FANCJ promotes DNA synthesis through G-quadruplex structures.

ABSTRACT: Our genome contains many G-rich sequences, which have the propensity to fold into stable secondary DNA structures called G4 or G-quadruplex structures. These structures have been implicated in cellular processes such as gene regulation and telomere maintenance. However, G4 sequences are prone to mutations particularly upon replication stress or in the absence of specific helicases. To investigate how G-quadruplex structures are resolved during DNA replication, we developed a model system using ssDNA templates and Xenopus egg extracts that recapitulates eukaryotic G4 replication. Here, we show that G-quadruplex structures form a barrier for DNA replication. Nascent strand synthesis is blocked at one or two nucleotides from the G4. After transient stalling, G-quadruplexes are efficiently unwound and replicated. In contrast, depletion of the FANCJ/BRIP1 helicase causes persistent replication stalling at G-quadruplex structures, demonstrating a vital role for this helicase in resolving these structures. FANCJ performs this function independently of the classical Fanconi anemia pathway. These data provide evidence that the G4 sequence instability in FANCJ(-/-) cells and Fancj/dog1 deficient C. elegans is caused by replication stalling at G-quadruplexes.

SUBMITTER: Castillo Bosch P

PROVIDER: S-EPMC4282361 | biostudies-literature |

REPOSITORIES: biostudies-literature

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FANCJ coordinates two pathways that maintain epigenetic stability at G-quadruplex DNA.

Project description:We have previously reported that DT40 cells deficient in the Y-family polymerase REV1 are defective in replicating G-quadruplex DNA. In vivo this leads to uncoupling of DNA synthesis from redeposition of histones displaced ahead of the replication fork, which in turn leads to loss of transcriptional repression due to failure to recycle pre-existing repressive histone post-translational modifications. Here we report that a similar process can also affect transcriptionally active genes, leading to their deactivation. We use this finding to develop an assay based on loss of expression of a cell surface marker to monitor epigenetic instability at the level of single cells. This assay allows us to demonstrate G4 DNA motif-associated epigenetic instability in mutants of three helicases previously implicated in the unwinding of G-quadruplex structures, FANCJ, WRN and BLM. Transcriptional profiling of DT40 mutants reveals that FANCJ coordinates two independent mechanisms to maintain epigenetic stability near G4 DNA motifs that are dependent on either REV1 or on the WRN and BLM helicases, suggesting a model in which efficient in vivo replication of G-quadruplexes often requires the established 5'-3'-helicase activity of FANCJ acting in concert with either a specialized polymerase or helicase operating in the opposite polarity.

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DNA G-quadruplex structures mold the DNA methylome.

Project description:Control of DNA methylation level is critical for gene regulation, and the factors that govern hypomethylation at CpG islands (CGIs) are still being uncovered. Here, we provide evidence that G-quadruplex (G4) DNA secondary structures are genomic features that influence methylation at CGIs. We show that the presence of G4 structure is tightly associated with CGI hypomethylation in the human genome. Surprisingly, we find that these G4 sites are enriched for DNA methyltransferase 1 (DNMT1) occupancy, which is consistent with our biophysical observations that DNMT1 exhibits higher binding affinity for G4s as compared to duplex, hemi-methylated, or single-stranded DNA. The biochemical assays also show that the G4 structure itself, rather than sequence, inhibits DNMT1 enzymatic activity. Based on these data, we propose that G4 formation sequesters DNMT1 thereby protecting certain CGIs from methylation and inhibiting local methylation.

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G-quadruplex recognition and remodeling by the FANCJ helicase.

Project description:Guanine rich nucleic acid sequences can form G-quadruplex (G4) structures that interfere with DNA replication, repair and RNA transcription. The human FANCJ helicase contributes to maintaining genomic integrity by promoting DNA replication through G4-forming DNA regions. Here, we combined single-molecule and ensemble biochemical analysis to show that FANCJ possesses a G4-specific recognition site. Through this interaction, FANCJ targets G4-containing DNA where its helicase and G4-binding activities enable repeated rounds of stepwise G4-unfolding and refolding. In contrast to other G4-remodeling enzymes, FANCJ partially stabilizes the G-quadruplex. This would preserve the substrate for the REV1 translesion DNA synthesis polymerase to incorporate cytosine across from a replication-stalling G-quadruplex. The residues responsible for G-quadruplex recognition also participate in interaction with MLH1 mismatch-repair protein, suggesting that the FANCJ activity supporting replication and its participation in DNA interstrand crosslink repair and/or heteroduplex rejection are mutually exclusive. Our findings not only describe the mechanism by which FANCJ recognizes G-quadruplexes and mediates their stepwise unfolding, but also explain how FANCJ chooses between supporting DNA repair versus promoting DNA replication through G-rich sequences.

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G-quadruplex structures moulds the DNA methylome

Project description:Control of DNA methylation level is critical for gene regulation, and the factors that govern hypomethylation at CpG islands (CGIs) are still being uncovered. Here, we provide evidence that G-quadruplex (G4) DNA secondary structures are genomic features that influence methylation at CGIs. We show that the presence of G4 structure is tightly associated with CGI hypomethylation. Surprisingly, we find that these G4 sites are enriched for DNA methyltransferase 1 (DNMT1) occupancy, which is consistent with our biophysical observations that DNMT1 exhibits higher binding affinity for G4s as compared to duplex, hemi-methylated or single-stranded DNA. The biochemical assays also show that the G4 structure itself, rather than sequence, inhibits DNMT1 enzymatic activity. Based on these data, we propose that G4 formation sequesters DNMT1 thereby protecting certain CGIs from methylation and inhibiting local methylation.

2018-07-16 | GSE107690 | GEO

Assembly of a G-Quadruplex Repair Complex by the FANCJ DNA Helicase and the REV1 Polymerase.

Project description:The FANCJ helicase unfolds G-quadruplexes (G4s) in human cells to support DNA replication. This action is coupled to the recruitment of REV1 polymerase to synthesize DNA across from a guanine template. The precise mechanisms of these reactions remain unclear. While FANCJ binds to G4s with an AKKQ motif, it is not known whether this site recognizes damaged G4 structures. FANCJ also has a PIP-like (PCNA Interacting Protein) region that may recruit REV1 to G4s either directly or through interactions mediated by PCNA protein. In this work, we measured the affinities of a FANCJ AKKQ peptide for G4s formed by (TTAGGG)4 and (GGGT)4 using fluorescence spectroscopy and biolayer interferometry (BLI). The effects of 8-oxoguanine (8oxoG) on these interactions were tested at different positions. BLI assays were then performed with a FANCJ PIP to examine its recruitment of REV1 and PCNA. FANCJ AKKQ bound tightly to a TTA loop and was sequestered away from the 8oxoG. Reducing the loop length between guanine tetrads increased the affinity of the peptide for 8oxoG4s. FANCJ PIP targeted both REV1 and PCNA but favored interactions with the REV1 polymerase. The impact of these results on the remodeling of damaged G4 DNA is discussed herein.

| S-EPMC7017153 | biostudies-literature

DNA secondary structures: stability and function of G-quadruplex structures.

Project description:In addition to the canonical double helix, DNA can fold into various other inter- and intramolecular secondary structures. Although many such structures were long thought to be in vitro artefacts, bioinformatics demonstrates that DNA sequences capable of forming these structures are conserved throughout evolution, suggesting the existence of non-B-form DNA in vivo. In addition, genes whose products promote formation or resolution of these structures are found in diverse organisms, and a growing body of work suggests that the resolution of DNA secondary structures is critical for genome integrity. This Review focuses on emerging evidence relating to the characteristics of G-quadruplex structures and the possible influence of such structures on genomic stability and cellular processes, such as transcription.

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FANCJ helicase defective in Fanconia anemia and breast cancer unwinds G-quadruplex DNA to defend genomic stability.

Project description:FANCJ mutations are associated with breast cancer and genetically linked to the bone marrow disease Fanconi anemia (FA). The genomic instability of FA-J mutant cells suggests that FANCJ helicase functions in the replicational stress response. A putative helicase with sequence similarity to FANCJ in Caenorhabditis elegans (DOG-1) and mouse (RTEL) is required for poly(G) tract maintenance, suggesting its involvement in the resolution of alternate DNA structures that impede replication. Under physiological conditions, guanine-rich sequences spontaneously assemble into four-stranded structures (G quadruplexes [G4]) that influence genomic stability. FANCJ unwound G4 DNA substrates in an ATPase-dependent manner. FANCJ G4 unwinding is specific since another superfamily 2 helicase, RECQ1, failed to unwind all G4 substrates tested under conditions in which the helicase unwound duplex DNA. Replication protein A stimulated FANCJ G4 unwinding, whereas the mismatch repair complex MSH2/MSH6 inhibited this activity. FANCJ-depleted cells treated with the G4-interactive compound telomestatin displayed impaired proliferation and elevated levels of apoptosis and DNA damage compared to small interfering RNA control cells, suggesting that G4 DNA is a physiological substrate of FANCJ. Although the FA pathway has been classically described in terms of interstrand cross-link (ICL) repair, the cellular defects associated with FANCJ mutation extend beyond the reduced ability to repair ICLs and involve other types of DNA structural roadblocks to replication.

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Pif1 is essential for efficient replisome progression through lagging strand G-quadruplex DNA secondary structures.

Project description:Pif1 DNA helicase is a potent unwinder of G-quadruplex (G4) structures in vitro and functions to maintain genome stability at G4 sequences in Saccharomyces cerevisiae. Here, we developed and utilized a live-cell imaging approach to quantitatively measure the progression rates of single replication forks through different G4 containing sequences in individual yeast cells. We show that in the absence of Pif1, replication rates through specific lagging strand G4 sequences in vivo is significantly decreased. In contrast, we found that in the absence of Pif1, replication rates through the same G4s on the leading strand are not decreased relative to the respective WT strains, showing that Pif1 is essential only for efficient replication through lagging strand G4s. Additionally, we show that a canonical PIP sequence in Pif1 interacts with PCNA and that replication through G4 structures is significantly slower in the absence of this interaction in vitro and in vivo. Thus, Pif1-PCNA interaction is essential for optimal replisome progression through G4 sequences, highlighting the importance of coupling between Pif1 activity and replisome progression during yeast genome replication.

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G-Quadruplex structures are stable and detectable in human genomic DNA.

Project description:The G-quadruplex is an alternative DNA structural motif that is considered to be functionally important in the mammalian genome. Herein, we address the hypothesis that G-quadruplex structures can exist within double-stranded genomic DNA using a G-quadruplex-specific probe. An engineered antibody is employed to enrich for DNA containing G-quadruplex structures, followed by deep sequencing to detect and map G-quadruplexes at high resolution in genomic DNA from human breast adenocarcinoma cells. Our high sensitivity structure-based pull-down strategy enables the isolation of genomic DNA fragments bearing a single as well as multiple G-quadruplex structures. Stable G-quadruplex structures are found in sub-telomeres, gene bodies and gene regulatory regions. For a sample of identified target genes, we show that G-quadruplex stabilizing ligands can modulate transcription. These results confirm the existence of G-quadruplex structures and their persistence in human genomic DNA. Four independent libraries have been enriched in DNA G-quadruplex structures using a G-quadruplex-specific probe. One genomic input library was sequenced as control. Deep-sequencing of these libraries allowed the mapping of G-quadruplexes on the genome.

2013-05-08 | E-GEOD-45241 | biostudies-arrayexpress

FANCJ helicase promotes DNA end resection by facilitating CtIP recruitment to DNA double-strand breaks.

Project description:FANCJ helicase mutations are known to cause hereditary breast and ovarian cancers as well as bone marrow failure syndrome Fanconi anemia. FANCJ plays an important role in the repair of DNA inter-strand crosslinks and DNA double-strand breaks (DSBs) by homologous recombination (HR). Nonetheless, the molecular mechanism by which FANCJ controls HR mediated DSB repair is obscure. Here, we show that FANCJ promotes DNA end resection by recruiting CtIP to the sites of DSBs. This recruitment of CtIP is dependent on FANCJ K1249 acetylation. Notably, FANCJ acetylation is dependent on FANCJ S990 phosphorylation by CDK. The CDK mediated phosphorylation of FANCJ independently facilitates its interaction with BRCA1 at damaged DNA sites and promotes DNA end resection by CtIP recruitment. Strikingly, mutational studies reveal that ATP binding competent but hydrolysis deficient FANCJ partially supports end resection, indicating that in addition to the scaffolding role of FANCJ in CtIP recruitment, its helicase activity is important for promoting end resection. Together, these data unravel a novel function of FANCJ helicase in DNA end resection and provide mechanistic insights into its role in repairing DSBs by HR and in genome maintenance.

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