Castillo Bosch P et al. (2014), FANCJ promotes DNA synthesis through G-quadrupl...

XB-ART-49425

EMBO J 2014 Nov 03;3321:2521-33. doi: 10.15252/embj.201488663.

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FANCJ promotes DNA synthesis through G-quadruplex structures.

Castillo Bosch P , Segura-Bayona S , Koole W , van Heteren JT , Dewar JM , Tijsterman M , Knipscheer P .

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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.

???displayArticle.pubmedLink??? 25193968
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Species referenced: Xenopus laevis
Genes referenced: brip1 c8h6orf47 fancd2
GO keywords: regulation of DNA biosynthetic process

???displayArticle.disOnts??? Fanconi anemia complementation group J
???displayArticle.omims??? FANCONI ANEMIA, COMPLEMENTATION GROUP J; FANCJ

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	Figure 1. G-quadruplex structures and sequencesAâG4 consensus sequence consisting of four stretches of at least three guanines (G) separated by 1â7 random nucleotides (N).BâSchematic representation of an antiparallel (left) and a parallel (right) G-quadruplex structure. G-planes stabilized by non-canonical Hoogsteen hydrogen bonds are shown in blue.CâG4 sequences and non-G4 control sequences used in this study.
	Figure 2. G-quadruplex structures block DNA synthesis by T7 DNA polymeraseAâSchematic representation of the primer extension assay. Extension is initiated from 32P-labeled (asterisk) primer B annealed to single-stranded pBluescript DNA containing a G4 sequence (left). If extension is blocked by the G-quadruplex structure, accumulation of a Ë200-nt product is expected (middle) while full extension generates a Ë3,000-nt product (right).BâG4 and non-G4 plasmid templates were subjected to primer extension by a modified T7 DNA polymerase (Sequenase). Extension was stopped after the indicated times, and reaction products were separated on 6% urea-PAGE gels and visualized by autoradiography.CâThe extension stalling products after 1Â min (from B) were quantified using ImageQuant software, and the percentage of this product versus the total of products that have arrived or bypassed the G4 sequence is depicted for the various sequences used.
	Figure 3. G-quadruplex structures are efficiently replicated in Xenopus egg extractsA Schematic representation of the G4 plasmid replication assay in HSS. Replication is started from primer A located 760 nucleotides (nt) from the G-quadruplex. Stalling of replication at the G-quadruplex structure will result in the accumulation of a 760-nt product, while G4 bypass first generates a 3,000-nt product that over time is ligated and supercoiled.BâG4G3N and non-G4C3p plasmid templates were replicated in HSS. Samples collected at the indicated times were separated on agarose gels and visualized with SybrGold (top panels). Gels were subsequently dried and visualized by autoradiography (bottom panels). The bands corresponding to the ssDNA, fully replicated but still nicked, and supercoiled dsDNA are indicated.CâNon-G4C3p, G4G3N, G4G5N, G4G15, and G4G23 plasmids were replicated in HSS, samples were taken at the indicated times, separated on 6% urea-PAGE gels and visualized by autoradiography. Products stalled at the G4 sequence (âstalledâ), linear molecules resulting from denatured nicked products (âlinearâ), and closed supercoiled products (âsupercoiledâ) are indicated.DâNon-G4C3p, G4G3N, G4G5N, G4G15, and G4G23 plasmid templates were replicated in HSS in the presence of 5Â Î¼M of Phen-DC3. Samples collected at the indicated times were separated on urea-PAGE gels and visualized by autoradiography.
	Figure 4. Mapping the sites of replication stalling at G-quadruplex structuresAâSchematic representation of a G4 template showing AseI restriction sites. Products formed after AseI digestion and/or replication stalling at the G-quadruplex structure are depicted on the right. Of note, AseI will only cut in double-stranded DNA and thus replication past the site is required.BâG4G5N and non-G4C3P plasmids were replicated in HSS. Samples collected at the indicated times were extracted, AseI digested, separated on a high-resolution urea-PAGE sequencing gel and visualized by autoradiography (top). Sequencing ladder generated by extension of primer A on pBluescript allows the mapping of the replication products. Stalling positions are depicted on top of the G4G5N sequence (bottom) and are numbered such that the first nucleotide within the G4 sequence is numbered 0, the last nucleotide 3â² to the G4 sequence is numbered -1 and so forth.C, D G4G3N (C) and G4G23 (D) plasmids were replicated in HSS, in the presence or absence of Phen-DC3, and analyzed as in (B). The sections of the sequencing gels containing the stalled replication products are depicted. Replication stalling sites were mapped and depicted on the relevant G4 sequences below the gels. Stalling positions were numbered as in (B).
	Figure 5. Depletion of FANCJ results in persistent stalling at G-quadruplex structuresAâMock-depleted, FANCJ-depleted, and FANCJ-depleted HSS supplemented with recombinant xlFANCJ were analyzed by Western blotting using xlFANCJ antibody. A dilution series of undepleted extract was loaded on the same blot to determine the degree of depletion. A relative volume of 100 corresponds to 0.7Â Î¼l of HSS. A non-specific band cross-reacting with FANCJ antibody is used as a loading control (âLoadingâ).BâMock-depleted, FANCJ-depleted, and FANCJ-depleted HSS supplemented with recombinant xlFANCJ were used to replicate the G4G3N plasmid template starting from primer A. Replication products were extracted, separated on 6% urea-PAGE gels, and visualized by autoradiography. Products stalled at the G4 sequence (âstalledâ), linear molecules resulting from denatured nicked products (âlinearâ), and closed supercoiled products (âsupercoiledâ) are indicated.CâG4G3N was replicated in FANCJ-depleted HSS starting from primer A. After 60Â min, xlFANCJ or buffer was added followed by an additional 30-min incubation. Replication products were extracted, separated on 6% urea-PAGE gels, and visualized by autoradiography.DâMock-depleted and FANCJ-depleted HSS were used to replicate G4G3N starting from primer A in the absence or presence of a low concentration (0.75Â Î¼M) of Phen-DC3. Replication products were extracted, separated on 6% urea-PAGE gels, and visualized by autoradiography.Eâ The G4 stalled and bypassed products of the 90-min time point in (D) were quantified using ImageQuant software, and the percentage of stalling versus bypassed products was plotted for the various conditions. Black bars represent percentage of stalling in the mock-depleted samples, the sum of black and dashed bars represent the percentage of stalling in the FANCJ-depleted samples. Therefore, dashed bars represent percentage of stalling as a result of FANCJ depletion only.
	Figure 6. Role of FANCJ in promoting G-quadruplex structure replication is independent of FANCD2AâG4G3N and non-G4C3p plasmids were replicated in HSS. Samples were taken at various time points and analyzed by Western blotting using xlFANCD2 antibody.BâMock-depleted and FANCD2-depleted HSS were analyzed by Western blotting using xlFANCJ and xlFANCD2 antibodies. A dilution series of undepleted extract was loaded on the same blot to determine the degree of depletion. A relative volume of 100 corresponds to 0.7Â Î¼l of HSS. A non-specific band cross-reacting with FANCJ antibody is used as loading control (âLoadingâ).CâMock-depleted and FANCD2-depleted HSS from (B) were used to replicate the G4G3N template starting from primer A. Replication products were extracted, separated on 6% urea-PAGE gels, and visualized by autoradiography.
	Figure 7. Model for G4 DNA recognition and unwinding during DNA replication(A) When the DNA replication machinery encounters a G-quadruplex structure, it temporarily stalls right at the G4 site (G4 detection). Stable G-quadruplex structures depend on FANCJ for their resolving (B), while others are resolved through FANCJ-independent mechanisms (D) after which DNA synthesis proceeds and the G4 sequence is faithfully replicated. In the absence of FANCJ (C), the G-quadruplex structures are not resolved leading to persistent replication stalling at the G4 sequence.

References [+] :

Aguilera, Causes of genome instability. 2013, Pubmed