Fig 2: Model for the interplay between ATRX and RECQ5 to regulate distinct subpathways of HR. Repair of two-ended DSBs by ATRX-mediated HR outcompetes RECQ5, possibly through PCNA binding, thereby suppressing SDSA. Upon the completion of DNA repair synthesis, the resulting JM is not subject to dissolution by BLM and is channelled into the resolution pathway by MUS81 and GEN1, leading to equal probability of CO and non-CO products.

Fig 3: ATRX and RECQ5 differentially affect long tract gene conversion and SCE formation. (A) U2OSATRX cells, with and without doxycycline-induced ATRX expression and transfected with siCtrl or siRECQ5, were labeled with EdU, irradiated, and then incubated with BrdU for 8 h. BrdU foci representing DNA repair synthesis were enumerated in EdU-negative G2 cells. Spontaneous foci (fewer than one) were subtracted. (B) Schematic diagram of the LTGC/STGC reporter system. U2OS cells carry a 5′ truncated GFP cassette followed by two incorrectly oriented exons (exon B placed 5′ to exon A) of the Blasticidin resistance (BsdR) gene, and a GFP cassette that is disrupted by the recognition sequence of the I-SceI endonuclease. To obtain a functional GFP protein, the break has to be repaired by HR using the truncated GFP cassette as a template. STGC events of fewer than 1 kbp result in a functional GFP gene but fail to correctly orient the exons of the BsdR gene, and the cells are thus sensitive to Blasticidin. LTGC events of >1 kbp restore the GFP sequence and additionally place exon B of the BsdR gene 3′ to exon A, reinstating correct orientation and conferring Blasticidin resistance. Thus, LTGC events lead to GFP+, BsdR+ cells [image modified from Nagaraju et al. (45)]. For the analysis, GFP+ cells (representing both STGC and LTGC events) and BsdR+ cells (LTGC events) are enumerated. (C) U2OS STGC/LTGC reporter cells were transfected with siCtrl or siRECQ5 and pUC19 or myc-ATRX and 40 h later transfected with I-SceI plasmids. Cells were assessed for GFP expression and Blasticidin resistance and numbers were plotted as a fraction of all cells. Control experiments showed that the fraction of S/G2-phase cells varied by less than 5% between siRECQ5-treated, ATRX-expressing, and control cells, excluding that changes in cell cycle distributions substantially influence the results. Knockdown was confirmed by immunoblotting and myc-ATRX expression efficiency was previously assessed (26). The significance indication above the white bar compares the LTGC fraction of pUC19 to the ATRX-expressing siCtrl-treated cells; indications above the black bars compare STGC fractions upon RECQ5 depletion to the respective siCtrl-treated cells. (D) U2OSATRX cells, with and without doxycycline-induced ATRX expression, were transfected with siCtrl or siRECQ5 and incubated with BrdU for 48 h. Cells were then irradiated with 2 Gy, collected after 16 h, and processed to obtain mitotic spreads. SCEs per spread were quantified and normalized to 70 chromosomes. Representative images of chromosome spreads of nonirradiated and irradiated siCtrl-treated cells with and without ATRX expression are shown; red arrows in the magnifications show individual SCE events. Data in A and C show mean ± SEM (n = 3). Results from individual experiments, each derived from 40 cells in A, are indicated. Individual SCE data are shown and red horizontal lines indicate the mean; 70 to 120 spreads and >4,000 chromosomes per condition were analyzed from three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001; ns: not significant (two-tailed t test); NIR: nonirradiated.

Fig 4: Loss of RECQ5 induces the RNA/DNA hybrid at rDNA loci. (A) Loss of RECQ5 leads to the accumulation of unprocessed pre-rRNA in FC. Nascent pre-rRNA was detected in nucleolus by 5′ETS probe in cells lacking RECQ5. SIM was performed. The staining of POLR1A indicates FC. Scale bars, 1 μm. The relative signal intensity of 5′ETS was calculated in 100 FC (right panel). (B) A model of RECQ5-mediated pre-rRNA releasing from FC. (C) Loss of RECQ5 induces accumulation of the RNA/DNA hybrid in nucleolus. The RNA/DNA hybrids were stained in nucleolus by S9.6 antibody. The staining of POLR1A indicates FC. SIM was performed. 100 nucleoli were examined in each group. Scale bars, 1 μm. The fluorescence intensity on the white dash line was plotted (lower right panel). (D) Ectopically expressed RNaseH1 suppresses the RNA/DNA hybrid. RNaseH1-GFP and empty vector were expressed in HeLa cells lacking RECQ5. The RNA/DNA hybrid was examined in nucleolus by S9.6. 100 nucleoli were examined in each group. Scale bars, 1 μm. (E) The positions of primer pairs at rDNA locus. (F) Loss of RECQ5 causes the RNA/DNA hybrid at rDNA locus. DRIP-qPCR was performed with S9.6 antibody and indicated primers. The assays were triplicated. (G) Wild-type but not cancer-associated mutations of RECQ5 suppress the RNA/DNA hybrid at rDNA locus. Data are represented as means ± SD as indicated. Two-tailed Student’s t-test was used to determine statistical significance. ***P < .001; ns, not significant.

Fig 5: Characterization of cancer-associated mutations of RECQ5. (A) Schematic of the representative mutations of RECQ5. (B) The truncation mutants, including Q139*, W334*, and S727Pfs*27, do not localize in nucleolus. GFP-tagged RECQ5 mutants and POLR1A were examined by IF. Scale bar, 10 μm. (C) The missense mutations, including S59F, P78L, and S102L, do not affect the nucleolar localization of RECQ5. The GFP-tagged RECQ5 mutants and POLR1A were examined in nucleolus by SIM. The fluorescence intensity on the white dash line was plotted (lower panels). (D) The missense mutations impair the helicase activities of RECQ5. Wild-type RECQ5 or RECQ5 mutants were incubated with the dsRNA substrates, and in vitro helicase assays were conducted. The reaction products were analyzed using gel electrophoresis (lower panels).