Padayachy L et al. (2024), RECQL4 is not critical for firing of human DNA ...

XB-ART-60652

Sci Rep 2024 Apr 02;141:7708. doi: 10.1038/s41598-024-58404-0.

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RECQL4 is not critical for firing of human DNA replication origins.

Padayachy L , Ntallis SG , Halazonetis TD .

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Human RECQL4, a member of the RecQ helicase family, plays a role in maintaining genomic stability, but its precise function remains unclear. The N-terminus of RECQL4 has similarity to Sld2, a protein required for the firing of DNA replication origins in budding yeast. Consistent with this sequence similarity, the Xenopus laevis homolog of RECQL4 has been implicated in initiating DNA replication in egg extracts. To determine whether human RECQL4 is required for firing of DNA replication origins, we generated cells in which both RECQL4 alleles were targeted, resulting in either lack of protein expression (knock-out; KO) or expression of a full-length, mutant protein lacking helicase activity (helicase-dead; HD). Interestingly, both the RECQL4 KO and HD cells were viable and exhibited essentially identical origin firing profiles as the parental cells. Analysis of the rate of fork progression revealed increased rates in the RECQL4 KO cells, which might be indicative of decreased origin firing efficiency. Our results are consistent with human RECQL4 having a less critical role in firing of DNA replication origins, than its budding yeast homolog Sld2.

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Species referenced: Xenopus laevis
Genes referenced: ctrl pcna recql4 ticrr
GO keywords: DNA replication

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	Figure 1\| RECQL4 is not critical for entry into S phase. (a) Immunoblot showing depletion of RECQL4 by specific siRNA (si) with PCNA serving as a loading control (top) and flow cytometry analysis of EdU incorporation in asynchronous cells following depletion of TRESLIN or RECQL4 (bottom). Ctrl, control; 2C and 4C, genomic DNA content. (b) Graphical representation of the human RECQL4 protein showing its three major protein domains and the sites targeted for mutagenesis. Red and green arrows, sites targeted in the KO and HD clones, respectively. fsH and fsN, frameshift mutations in the Helicase and N-terminal domains, respectively; KM, lysine to methionine substitution in the helicase domain. (c) Immunoblot analysis showing the levels of RECQL4 expression in the KO and HD clones and induction of Cyclin E. PCNA and Lamin A serve as loading controls. NE, normal levels of Cyclin E; OE, overexpression of Cyclin E. (d) Experimental setup (top) and kinetics of S-phase entry of parental cells and RECQL4-mutant clones, as ascertained by flow cytometry-based analysis of EdU-positive cells (% EdU +) at different time points after mitotic shake-off (bottom). Averages and standard deviations from three independent experiments are shown. Clone fsH2 entered S phase faster than the parental cells, when Cyclin E was overexpressed (P < 0.001).
	Figure 2\| Targeting RECQL4 has no effect on DNA replication origin firing profiles. (a) Experimental setup for EdU-seq. (b) and (d) Overlay of DNA replication origin firing profiles of parental cells (WT) and RECQL4-mutant clones for a representative genomic region under conditions of normal Cyclin E expression (NE) (b) or Cyclin E overexpression (OE) (d). The EdU-seq data are presented as sigma (σ) values. RT; replication timing (blue, early; green, mid S phase); Ge, genes (green, forward direction of transcription; red, reverse; yellow, unspecified; blue, multiple genes within bin); iG, intergenic regions (gray). Bin resolution: 10 kb. (c) and (e) Correlation plots of the sigma values of all constitutive (CN, purple) and oncogene-induced (Oi, red) origins of the parental cells versus the RECQL4-mutant clones under conditions of normal Cyclin E expression (c) or Cyclin E overexpression (e).
	Figure 3\| RECQL4 affects DNA replication fork progression. (a) Experimental outline for the analysis of fork progression by DNA combing in synchronized cells. (b) and (c) Lengths of IdU tracks of parental cells (WT) and RECQL4-mutant clones treated as shown in (a). NE, normal levels of Cyclin E (b); OE, Cyclin E overexpression (c). More than 190 IdU tracks were measured per sample. (d) Experimental outline for the analysis of fork progression by DNA combing in asynchronous cells. (e) and (f) Lengths of CldU (e) and IdU (f) tracks of parental cells (WT) and RECQL4-mutant clones treated according to the outline shown in (d). The cells expressed normal levels of Cyclin E (NE). More than 150 CldU-IdU double-labeled fibers were measured per sample. For all samples, the median and upper and lower quartiles are indicated by horizontal lines. P values were calculated by a two-way ANOVA with Fisher’s Least Significance Difference test. , P < 0.05; ***, P < 0.0001; ns: not significant. (g) Experimental outline for the study of fork progression by EdU-seq. For the 0 min (’ or min) timepoint, after mitotic shake-off, the cells were incubated with HU and EdU for 14 h. (h) Genome-wide averages of the EdU-seq sigma values of the indicated samples and timepoints over genomic regions spanning 0.6 Mb around origins of replication. NE, normal levels of Cyclin E; OE, overexpression of Cyclin E; aσ, adjusted sigma values relative to the no-release (0 min) samples.
	Figure 4 \| RECQL4 is not critical for MiDAS. (a) Experimental outline for the assessment of the presence of MiDAS in mitotic parental (WT) and RECQL4 mutant cells by epifluorescence microscopy. (b) Fraction of EdU + mitotic cells, according to (a); at least two EdU foci per mitosis were required to consider a cell positive for MiDAS. Means and standard deviation of three independent experiments are shown on the graph. At least 130 mitotic cells were analyzed for each condition. Statistical analysis was performed with 2-way ANOVA with Tukey’s Multiple Comparisons test. ns, not significant. (c) Experimental outline for the study of mitotic DNA synthesis by MiDAS-seq. (d) Average MiDAS-seq signal (σ values) of all MiDAS regions and heatmap of the MiDAS-seq signal of each MiDAS region ranked according to its genomic size for single- or double-peak regions. Bin resolution: 10 kb. (e) Experimental outline for monitoring 53BP1 nuclear bodies in G1. (f) Fraction of cells containing 53BP1 nuclear bodies in early G1, according to (e). The graph shows averages and standard deviations from three independent experiments, with at least 230 cells analyzed per cell line. No significant differences between the parental cells and RECQL4 mutant clones were observed (2-way ANOVA with Tukey’s Multiple Comparisons test). Aph, aphidicolin; RO, RO3306.

References [+] :

Abe, The N-terminal region of RECQL4 lacking the helicase domain is both essential and sufficient for the viability of vertebrate cells. Role of the N-terminal region of RECQL4 in cells. 2011, Pubmed
Bartkova, DNA damage response as a candidate anti-cancer barrier in early human tumorigenesis. 2005, Pubmed
Boos, Origin Firing Regulations to Control Genome Replication Timing. 2019, Pubmed
Boos, Identification of a heteromeric complex that promotes DNA replication origin firing in human cells. 2013, Pubmed
Boos, Regulation of DNA replication through Sld3-Dpb11 interaction is conserved from yeast to humans. 2011, Pubmed
Chang, The nonsense-mediated decay RNA surveillance pathway. 2007, Pubmed
Costantino, Break-induced replication repair of damaged forks induces genomic duplications in human cells. 2014, Pubmed
Crevel, Drosophila RecQ4 is directly involved in both DNA replication and the response to UV damage in S2 cells. 2012, Pubmed
Croteau, Human RecQ helicases in DNA repair, recombination, and replication. 2014, Pubmed
Cvetkovic, The structural mechanism of dimeric DONSON in replicative helicase activation. 2023, Pubmed , Xenbase
Deegan, Phosphopeptide binding by Sld3 links Dbf4-dependent kinase to MCM replicative helicase activation. 2016, Pubmed
Dewar, The mechanism of DNA replication termination in vertebrates. 2015, Pubmed , Xenbase
Evrin, DONSON is required for CMG helicase assembly in the mammalian cell cycle. 2023, Pubmed
Fang, Sld3-MCM Interaction Facilitated by Dbf4-Dependent Kinase Defines an Essential Step in Eukaryotic DNA Replication Initiation. 2016, Pubmed
Fragkos, DNA replication origin activation in space and time. 2015, Pubmed
Gaggioli, CDK phosphorylation of SLD-2 is required for replication initiation and germline development in C. elegans. 2014, Pubmed
Garcia, Identification and functional analysis of TopBP1 and its homologs. 2005, Pubmed
Glover, DNA polymerase alpha inhibition by aphidicolin induces gaps and breaks at common fragile sites in human chromosomes. 1984, Pubmed
Harrigan, Replication stress induces 53BP1-containing OPT domains in G1 cells. 2011, Pubmed
Hashimoto, Novel role of DONSON in CMG helicase assembly during vertebrate DNA replication initiation. 2023, Pubmed , Xenbase
Ji, Genome-wide high-resolution mapping of mitotic DNA synthesis sites and common fragile sites by direct sequencing. 2020, Pubmed
Kingsley, DONSON facilitates Cdc45 and GINS chromatin association and is essential for DNA replication initiation. 2023, Pubmed , Xenbase
Kumagai, MTBP, the partner of Treslin, contains a novel DNA-binding domain that is essential for proper initiation of DNA replication. 2017, Pubmed , Xenbase
Kumagai, Direct regulation of Treslin by cyclin-dependent kinase is essential for the onset of DNA replication. 2011, Pubmed , Xenbase
Kumagai, Treslin collaborates with TopBP1 in triggering the initiation of DNA replication. 2010, Pubmed , Xenbase
Lim, In silico protein interaction screening uncovers DONSON's role in replication initiation. 2023, Pubmed , Xenbase
Lukas, 53BP1 nuclear bodies form around DNA lesions generated by mitotic transmission of chromosomes under replication stress. 2011, Pubmed
Macheret, Intragenic origins due to short G1 phases underlie oncogene-induced DNA replication stress. 2018, Pubmed
Macheret, Monitoring early S-phase origin firing and replication fork movement by sequencing nascent DNA from synchronized cells. 2019, Pubmed
Macheret, High-resolution mapping of mitotic DNA synthesis regions and common fragile sites in the human genome through direct sequencing. 2020, Pubmed
Mah, gammaH2AX: a sensitive molecular marker of DNA damage and repair. 2010, Pubmed
Marino, Bioinformatic analysis of RecQ4 helicases reveals the presence of a RQC domain and a Zn knuckle. 2013, Pubmed
Matsuno, The N-terminal noncatalytic region of Xenopus RecQ4 is required for chromatin binding of DNA polymerase alpha in the initiation of DNA replication. 2006, Pubmed , Xenbase
McFarland, Improved estimation of cancer dependencies from large-scale RNAi screens using model-based normalization and data integration. 2018, Pubmed
Minocherhomji, Replication stress activates DNA repair synthesis in mitosis. 2015, Pubmed
Mojumdar, The Human RecQ4 Helicase Contains a Functional RecQ C-terminal Region (RQC) That Is Essential for Activity. 2017, Pubmed
Mueller, DNA replication: mammalian Treslin-TopBP1 interaction mirrors yeast Sld3-Dpb11. 2011, Pubmed
Muramatsu, CDK-dependent complex formation between replication proteins Dpb11, Sld2, Pol (epsilon}, and GINS in budding yeast. 2010, Pubmed
Oughtred, The BioGRID database: A comprehensive biomedical resource of curated protein, genetic, and chemical interactions. 2021, Pubmed
Poli, dNTP pools determine fork progression and origin usage under replication stress. 2012, Pubmed
Rogakou, DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139. 1998, Pubmed
Rossi, Conserved helicase domain of human RecQ4 is required for strand annealing-independent DNA unwinding. 2010, Pubmed
Sanchez-Pulido, Homology explains the functional similarities of Treslin/Ticrr and Sld3. 2010, Pubmed
Sangrithi, Initiation of DNA replication requires the RECQL4 protein mutated in Rothmund-Thomson syndrome. 2005, Pubmed , Xenbase
Sansam, A vertebrate gene, ticrr, is an essential checkpoint and replication regulator. 2010, Pubmed
Schindelin, Fiji: an open-source platform for biological-image analysis. 2012, Pubmed
Schultz, p53 binding protein 1 (53BP1) is an early participant in the cellular response to DNA double-strand breaks. 2000, Pubmed
Smeets, The Rothmund-Thomson syndrome helicase RECQL4 is essential for hematopoiesis. 2014, Pubmed
Tanaka, CDK-dependent phosphorylation of Sld2 and Sld3 initiates DNA replication in budding yeast. 2007, Pubmed
Thangavel, Human RECQ1 and RECQ4 helicases play distinct roles in DNA replication initiation. 2010, Pubmed
Tognetti, Switch on the engine: how the eukaryotic replicative helicase MCM2-7 becomes activated. 2015, Pubmed
Tourrière, Single-molecule Analysis of DNA Replication Dynamics in Budding Yeast and Human Cells by DNA Combing. 2017, Pubmed
Tsherniak, Defining a Cancer Dependency Map. 2017, Pubmed
Técher, The impact of replication stress on replication dynamics and DNA damage in vertebrate cells. 2017, Pubmed
Volpi, The role of DDK and Treslin-MTBP in coordinating replication licensing and pre-initiation complex formation. 2021, Pubmed , Xenbase
Wu, Drosophila homologue of the Rothmund-Thomson syndrome gene: essential function in DNA replication during development. 2008, Pubmed , Xenbase
Xia, DNSN-1 recruits GINS for CMG helicase assembly during DNA replication initiation in Caenorhabditis elegans. 2023, Pubmed
Xu, dRecQ4 is required for DNA synthesis and essential for cell proliferation in Drosophila. 2009, Pubmed
Yeeles, Regulated eukaryotic DNA replication origin firing with purified proteins. 2015, Pubmed
Zegerman, Phosphorylation of Sld2 and Sld3 by cyclin-dependent kinases promotes DNA replication in budding yeast. 2007, Pubmed
Zegerman, Evolutionary conservation of the CDK targets in eukaryotic DNA replication initiation. 2015, Pubmed
Zhong, The level of origin firing inversely affects the rate of replication fork progression. 2013, Pubmed

	Figure 1\| RECQL4 is not critical for entry into S phase. (a) Immunoblot showing depletion of RECQL4 by specific siRNA (si) with PCNA serving as a loading control (top) and flow cytometry analysis of EdU incorporation in asynchronous cells following depletion of TRESLIN or RECQL4 (bottom). Ctrl, control; 2C and 4C, genomic DNA content. (b) Graphical representation of the human RECQL4 protein showing its three major protein domains and the sites targeted for mutagenesis. Red and green arrows, sites targeted in the KO and HD clones, respectively. fsH and fsN, frameshift mutations in the Helicase and N-terminal domains, respectively; KM, lysine to methionine substitution in the helicase domain. (c) Immunoblot analysis showing the levels of RECQL4 expression in the KO and HD clones and induction of Cyclin E. PCNA and Lamin A serve as loading controls. NE, normal levels of Cyclin E; OE, overexpression of Cyclin E. (d) Experimental setup (top) and kinetics of S-phase entry of parental cells and RECQL4-mutant clones, as ascertained by flow cytometry-based analysis of EdU-positive cells (% EdU +) at different time points after mitotic shake-off (bottom). Averages and standard deviations from three independent experiments are shown. Clone fsH2 entered S phase faster than the parental cells, when Cyclin E was overexpressed (P < 0.001).
	Figure 2\| Targeting RECQL4 has no effect on DNA replication origin firing profiles. (a) Experimental setup for EdU-seq. (b) and (d) Overlay of DNA replication origin firing profiles of parental cells (WT) and RECQL4-mutant clones for a representative genomic region under conditions of normal Cyclin E expression (NE) (b) or Cyclin E overexpression (OE) (d). The EdU-seq data are presented as sigma (σ) values. RT; replication timing (blue, early; green, mid S phase); Ge, genes (green, forward direction of transcription; red, reverse; yellow, unspecified; blue, multiple genes within bin); iG, intergenic regions (gray). Bin resolution: 10 kb. (c) and (e) Correlation plots of the sigma values of all constitutive (CN, purple) and oncogene-induced (Oi, red) origins of the parental cells versus the RECQL4-mutant clones under conditions of normal Cyclin E expression (c) or Cyclin E overexpression (e).
	Figure 3\| RECQL4 affects DNA replication fork progression. (a) Experimental outline for the analysis of fork progression by DNA combing in synchronized cells. (b) and (c) Lengths of IdU tracks of parental cells (WT) and RECQL4-mutant clones treated as shown in (a). NE, normal levels of Cyclin E (b); OE, Cyclin E overexpression (c). More than 190 IdU tracks were measured per sample. (d) Experimental outline for the analysis of fork progression by DNA combing in asynchronous cells. (e) and (f) Lengths of CldU (e) and IdU (f) tracks of parental cells (WT) and RECQL4-mutant clones treated according to the outline shown in (d). The cells expressed normal levels of Cyclin E (NE). More than 150 CldU-IdU double-labeled fibers were measured per sample. For all samples, the median and upper and lower quartiles are indicated by horizontal lines. P values were calculated by a two-way ANOVA with Fisher’s Least Significance Difference test. , P < 0.05; ***, P < 0.0001; ns: not significant. (g) Experimental outline for the study of fork progression by EdU-seq. For the 0 min (’ or min) timepoint, after mitotic shake-off, the cells were incubated with HU and EdU for 14 h. (h) Genome-wide averages of the EdU-seq sigma values of the indicated samples and timepoints over genomic regions spanning 0.6 Mb around origins of replication. NE, normal levels of Cyclin E; OE, overexpression of Cyclin E; aσ, adjusted sigma values relative to the no-release (0 min) samples.
	Figure 4 \| RECQL4 is not critical for MiDAS. (a) Experimental outline for the assessment of the presence of MiDAS in mitotic parental (WT) and RECQL4 mutant cells by epifluorescence microscopy. (b) Fraction of EdU + mitotic cells, according to (a); at least two EdU foci per mitosis were required to consider a cell positive for MiDAS. Means and standard deviation of three independent experiments are shown on the graph. At least 130 mitotic cells were analyzed for each condition. Statistical analysis was performed with 2-way ANOVA with Tukey’s Multiple Comparisons test. ns, not significant. (c) Experimental outline for the study of mitotic DNA synthesis by MiDAS-seq. (d) Average MiDAS-seq signal (σ values) of all MiDAS regions and heatmap of the MiDAS-seq signal of each MiDAS region ranked according to its genomic size for single- or double-peak regions. Bin resolution: 10 kb. (e) Experimental outline for monitoring 53BP1 nuclear bodies in G1. (f) Fraction of cells containing 53BP1 nuclear bodies in early G1, according to (e). The graph shows averages and standard deviations from three independent experiments, with at least 230 cells analyzed per cell line. No significant differences between the parental cells and RECQL4 mutant clones were observed (2-way ANOVA with Tukey’s Multiple Comparisons test). Aph, aphidicolin; RO, RO3306.