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. 2013 Apr 22;12(1):29.
doi: 10.1186/1476-4598-12-29.

Replication stress induces specific enrichment of RECQ1 at common fragile sites FRA3B and FRA16D

Xing Lu  1 , Swetha Parvathaneni, Toshifumi Hara, Ashish Lal, Sudha Sharma
Affiliations

Replication stress induces specific enrichment of RECQ1 at common fragile sites FRA3B and FRA16D

Xing Lu et al. Mol Cancer. .

Abstract

Background: Stalled replication forks at common fragile sites are a major cause of genomic instability. RecQ helicases, a highly conserved family of DNA-unwinding enzymes, are believed to ease 'roadblocks' that pose challenge to replication fork progression. Among the five known RecQ homologs in humans, functions of RECQ1, the most abundant of all, are poorly understood. We previously determined that RECQ1 helicase preferentially binds and unwinds substrates that mimic DNA replication/repair intermediates, and interacts with proteins involved in DNA replication restart mechanisms.

Method: We have utilized chromatin immunoprecipitation followed by quantitative real-time PCR to investigate chromatin interactions of RECQ1 at defined genetic loci in the presence or absence of replication stress. We have also tested the sensitivity of RECQ1-depleted cells to aphidicolin induced replication stress.

Results: RECQ1 binds to the origins of replication in unperturbed cells. We now show that conditions of replication stress induce increased accumulation of RECQ1 at the lamin B2 origin in HeLa cells. Consistent with a role in promoting fork recovery or repair, RECQ1 is specifically enriched at two major fragile sites FRA3B and FRA16D where replication forks have stalled following aphidicolin treatment. RECQ1-depletion results in attenuated checkpoint activation in response to replication stress, increased sensitivity to aphidicolin and chromosomal instability.

Conclusions: Given a recent biochemical observation that RECQ1 catalyzes strand exchange on stalled replication fork structures in vitro, our results indicate that RECQ1 facilitates repair of stalled or collapsed replication forks and preserves genome integrity. Our findings provide the first evidence of a crucial role for RECQ1 at naturally occurring fork stalling sites and implicate RECQ1 in mechanisms underlying common fragile site instability in cancer.

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Figures

Figure 1
Figure 1
Enhanced enrichment of RECQ1 at lamin B2 origin after replication stress. A. Genomic regions containing the human lamin B2 origin of replication. Primer sets used for qPCR analyses of origin (B48) and non-origin (B13) containing DNA are indicated. B. Quantification of cross-linked lamin B2 origin immunoprecipitated using the indicated antibodies. HeLa cells were either untreated or incubated with APH (0.5 μM) or HU (2 mM) for 24 h and processed for ChIP using a RECQ1-specific antibody. ORC2 antibody was used as positive control for origin-enrichment, and rabbit IgG served as negative control. qPCR was performed with origin-specific and non-origin primers for the lamin B2 locus. Fold enrichment was determined over IgG and is shown for each primer pair. Results are expressed as means ± SEM for at least three independent experiments. Specificity of RECQ1 antibody in immunoprecipitation (IP) of endogenous RECQ1 from HeLa extract is shown; input represents 10% of the extract used for IP. C. A representative gel of the amplified DNA immunoprecipitated with indicated antibodies. Input represents 1% of the cross-linked chromatin used for ChIP. D. Cell cycle profiles of untreated and HU or APH treated (24 h) HeLa cells. Propidium iodide stained cells were analyzed by FACS for DNA content; distribution in the G1, S, and G2/M phases of the cell cycle is indicated. E. Total RECQ1 protein level in HeLa cells following 24 h treatment with HU (2 mM) or APH (0.5 μM). GAPDH is loading control. F. Replication stress-dependent chromatin association of RECQ1. Western blot detection of RECQ1 in chromatin-enriched fractions of untreated and APH (0.5 μM), HU (2 mM) or MMC (0.5 μg/ml) treated cells, or following pretreatment with APH for 4 h before incubation with MMC (16 h). Histone H3 is loading control and chromatin marker. APH, aphidicolin; HU, hydroxyurea; MMC, mitomycin C.
Figure 2
Figure 2
RECQ1 is recruited to common fragile site FRA3B after treatment with aphidicolin. A. Genomic organization of the FRA3B region. Primer sets used for qPCR analyses of distal (FDR) and central (FCR) region within the FRA3B locus are indicated. B. Quantification of cross-linked FRA3B chromatin immunoprecipitated from HeLa cells using the indicated antibodies. HeLa cells were either untreated or treated with aphidicolin (0.5 μM) for 24 h and then processed for ChIP using a RECQ1-specific antibody. Phosphorylated H2AX (γH2AX) antibody was used as a positive control for FRA3B enrichment, and rabbit IgG served as negative control in ChIP experiments. qPCR was performed with two different sets of primers specific for the central and distal regions within FRA3B locus. Fold enrichment over IgG was determined and is shown for each primer pair for the ChIP. Results are expressed as means ± SEM for at least three independent experiments. C. A representative gel of the amplified DNA immunoprecipitated with indicated antibodies. Input represents 1% of the cross-linked chromatin used for ChIP. D. Binding of RECQ1 to FRA3B after treatment with hydroxyurea. Quantification of cross-linked FRA3B chromatin immunoprecipitated from HeLa cells using the indicated antibodies. HeLa cells were either untreated or treated with hydroxyurea (2 mM) for 24 h and processed for ChIP using a RECQ1-specific antibody. γH2AX antibody was used as a positive control for FRA3B enrichment, and rabbit IgG served as negative control in ChIP experiments. qPCR was performed with two different sets of primers specific for the central and distal regions within FRA3B locus. Fold enrichment over IgG was determined and is shown for each primer pair for the ChIP. Results are expressed as means ± SEM for at least three independent experiments. APH, aphidicolin; HU, hydroxyurea.
Figure 3
Figure 3
RECQ1 preferentially binds to FRA16D after treatment with aphidicolin. A. Genomic organization of the FRA16D region. Primer set used for qPCR analyses of the FRA16D locus is indicated. B. Quantification of cross-linked FRA16D chromatin immunoprecipitated from HeLa cells untreated or treated with aphidicolin (0.5 μM) for 24 h using a specific RECQ1 antibody or rabbit IgG. Fold enrichment of FRA16D containing sequences in RECQ1 ChIP was determined by normalizing enrichment obtained with IgG and is shown for both untreated and APH-treated cells. Relative occupancy of RECQ1 at FRA16D versus a non-fragile negative control site GAPDH shows preferential recruitment of RECQ1 to fragile site locus in aphidicolin treated cells. Results are expressed as means ± SEM for at least three independent experiments. C. A representative gel of the amplified DNA immunoprecipitated with indicated antibodies. Input represents 1% of the cross-linked chromatin used for ChIP. D. qPCR analyses of RECQ1, ORC2 or γH2AX -binding in ChIP experiments to DNA sequence containing β-actin in HeLa cells. E. A representative gel of the amplified β-actin sequence immunoprecipitated with indicated antibodies. Input represents 1% of the cross-linked chromatin used for ChIP. APH, aphidicolin.
Figure 4
Figure 4
RECQ1-depletion leads to aphidicolin sensitivity, aberrant replication stress response and chromosomal fragility in HeLa cells. A. Cells transfected with control or RECQ1 siRNA were exposed in quadruplicate to increasing doses of aphidicolin (μM) or grown in regular complete medium and their survival was measured 72 h later by MTS assay. Percentage of control growth was plotted for each data point, representing the mean ± SD of three independent experiments. B. Surviving fraction was determined for control or RECQ1 siRNA transfected cells growing in regular medium or medium supplemented with aphidicolin (μM) by cell count after 72 h and presented as the mean ± SD of three independent experiments. C. Detection of the activated forms of Chk1 kinase and the phosphorylated forms of RPA32 and H2AX in untreated or aphidicolin (0.5 μM) treated HeLa cells transfected with control or RECQ1 siRNA. Depletion of RECQ1 by siRNA is also shown. GAPDH serves as loading control. DandE. Chromosomal breaks/gaps in RECQ1-depleted cells. Metaphase spreads of control or RECQ1 siRNA transected cells (72 h after siRNA) grown in the absence or presence of aphidicolin (0.5 μM, 24 h) were scored for chromosome gaps/breaks. Ten metaphases of each cell condition were analyzed. In untreated condition, RECQ1-depleted cells showed significantly more breaks than control cells (p < 0.05); aphidicolin treatment induced chromosome breaks and gaps in control or RECQ1 siRNA transfected cells (D). Representative partial metaphase spreads for RECQ1-depletion is shown (E); bottom panel shows an enlarged section. APH, aphidicolin; HU, hydroxyurea.
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