hDNA 2 nuclease/helicase promotes centromeric DNA replication and genome stability
2018; Springer Nature; Volume: 37; Issue: 14 Linguagem: Inglês
10.15252/embj.201796729
ISSN1460-2075
AutoresZhengke Li, Bochao Liu, Weiwei Jin, Xiwei Wu, Mian Zhou, Vincent Liu, Ajay Goel, Zhiyuan Shen, Li Zheng, Binghui Shen,
Tópico(s)Chromosomal and Genetic Variations
ResumoArticle17 May 2018Open Access Source DataTransparent process hDNA2 nuclease/helicase promotes centromeric DNA replication and genome stability Zhengke Li Zhengke Li Department of Cancer Genetics and Epigenetics, Beckman Research Institute, City of Hope, Duarte, CA, USA Search for more papers by this author Bochao Liu Bochao Liu Department of Radiation Oncology, Rutgers Cancer Institute of New Jersey, Rutgers Robert Wood Johnson Medical School, Rutgers, the State University of New Jersey, New Brunswick, NJ, USA Search for more papers by this author Weiwei Jin Weiwei Jin Department of Cancer Genetics and Epigenetics, Beckman Research Institute, City of Hope, Duarte, CA, USA Department of Gastroenterology & Pancreatic Surgery, Zhejiang Provincial People's Hospital, Hangzhou, Zhejiang, China Search for more papers by this author Xiwei Wu Xiwei Wu Department of Molecular and Cellular Biology, Beckman Research Institute, City of Hope, Duarte, CA, USA Search for more papers by this author Mian Zhou Mian Zhou Department of Cancer Genetics and Epigenetics, Beckman Research Institute, City of Hope, Duarte, CA, USA Search for more papers by this author Vincent Zewen Liu Vincent Zewen Liu Department of Cancer Genetics and Epigenetics, Beckman Research Institute, City of Hope, Duarte, CA, USA Department of Computer Science, Columbia University, New York, NY, USACorrection added online on 23 May 2018 after first online publication: the author name has been corrected. Search for more papers by this author Ajay Goel Ajay Goel Center for Gastrointestinal Research, Center for Translational Genomics and Oncology, Baylor Scott and White Research Institute and Charles A. Sammons Cancer Center, Baylor University Medical Center, Dallas, TX, USA Search for more papers by this author Zhiyuan Shen Zhiyuan Shen orcid.org/0000-0003-2834-0309 Department of Radiation Oncology, Rutgers Cancer Institute of New Jersey, Rutgers Robert Wood Johnson Medical School, Rutgers, the State University of New Jersey, New Brunswick, NJ, USA Search for more papers by this author Li Zheng Corresponding Author Li Zheng [email protected] orcid.org/0000-0002-3744-185X Department of Cancer Genetics and Epigenetics, Beckman Research Institute, City of Hope, Duarte, CA, USA Search for more papers by this author Binghui Shen Corresponding Author Binghui Shen [email protected] orcid.org/0000-0002-4408-407X Department of Cancer Genetics and Epigenetics, Beckman Research Institute, City of Hope, Duarte, CA, USA Search for more papers by this author Zhengke Li Zhengke Li Department of Cancer Genetics and Epigenetics, Beckman Research Institute, City of Hope, Duarte, CA, USA Search for more papers by this author Bochao Liu Bochao Liu Department of Radiation Oncology, Rutgers Cancer Institute of New Jersey, Rutgers Robert Wood Johnson Medical School, Rutgers, the State University of New Jersey, New Brunswick, NJ, USA Search for more papers by this author Weiwei Jin Weiwei Jin Department of Cancer Genetics and Epigenetics, Beckman Research Institute, City of Hope, Duarte, CA, USA Department of Gastroenterology & Pancreatic Surgery, Zhejiang Provincial People's Hospital, Hangzhou, Zhejiang, China Search for more papers by this author Xiwei Wu Xiwei Wu Department of Molecular and Cellular Biology, Beckman Research Institute, City of Hope, Duarte, CA, USA Search for more papers by this author Mian Zhou Mian Zhou Department of Cancer Genetics and Epigenetics, Beckman Research Institute, City of Hope, Duarte, CA, USA Search for more papers by this author Vincent Zewen Liu Vincent Zewen Liu Department of Cancer Genetics and Epigenetics, Beckman Research Institute, City of Hope, Duarte, CA, USA Department of Computer Science, Columbia University, New York, NY, USACorrection added online on 23 May 2018 after first online publication: the author name has been corrected. Search for more papers by this author Ajay Goel Ajay Goel Center for Gastrointestinal Research, Center for Translational Genomics and Oncology, Baylor Scott and White Research Institute and Charles A. Sammons Cancer Center, Baylor University Medical Center, Dallas, TX, USA Search for more papers by this author Zhiyuan Shen Zhiyuan Shen orcid.org/0000-0003-2834-0309 Department of Radiation Oncology, Rutgers Cancer Institute of New Jersey, Rutgers Robert Wood Johnson Medical School, Rutgers, the State University of New Jersey, New Brunswick, NJ, USA Search for more papers by this author Li Zheng Corresponding Author Li Zheng [email protected] orcid.org/0000-0002-3744-185X Department of Cancer Genetics and Epigenetics, Beckman Research Institute, City of Hope, Duarte, CA, USA Search for more papers by this author Binghui Shen Corresponding Author Binghui Shen [email protected] orcid.org/0000-0002-4408-407X Department of Cancer Genetics and Epigenetics, Beckman Research Institute, City of Hope, Duarte, CA, USA Search for more papers by this author Author Information Zhengke Li1, Bochao Liu2, Weiwei Jin1,3, Xiwei Wu4, Mian Zhou1, Vincent Zewen Liu1,5, Ajay Goel6, Zhiyuan Shen2, Li Zheng *,1 and Binghui Shen *,1 1Department of Cancer Genetics and Epigenetics, Beckman Research Institute, City of Hope, Duarte, CA, USA 2Department of Radiation Oncology, Rutgers Cancer Institute of New Jersey, Rutgers Robert Wood Johnson Medical School, Rutgers, the State University of New Jersey, New Brunswick, NJ, USA 3Department of Gastroenterology & Pancreatic Surgery, Zhejiang Provincial People's Hospital, Hangzhou, Zhejiang, China 4Department of Molecular and Cellular Biology, Beckman Research Institute, City of Hope, Duarte, CA, USA 5Department of Computer Science, Columbia University, New York, NY, USA 6Center for Gastrointestinal Research, Center for Translational Genomics and Oncology, Baylor Scott and White Research Institute and Charles A. Sammons Cancer Center, Baylor University Medical Center, Dallas, TX, USA *Corresponding author. Tel: +1 626 301 8879; Fax: +1 626 301 8892; E-mail: [email protected] *Corresponding author. Tel: +1 626 301 8879; Fax: +1 626 301 8892; E-mail: [email protected] The EMBO Journal (2018)37:e96729https://doi.org/10.15252/embj.201796729 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract DNA2 is a nuclease/helicase that is involved in Okazaki fragment maturation, replication fork processing, and end resection of DNA double-strand breaks. Similar such helicase activity for resolving secondary structures and structure-specific nuclease activity are needed during DNA replication to process the chromosome-specific higher order repeat units present in the centromeres of human chromosomes. Here, we show that DNA2 binds preferentially to centromeric DNA. The nuclease and helicase activities of DNA2 are both essential for resolution of DNA structural obstacles to facilitate DNA replication fork movement. Loss of DNA2-mediated clean-up mechanisms impairs centromeric DNA replication and CENP-A deposition, leading to activation of the ATR DNA damage checkpoints at centromeric DNA regions and late-S/G2 cell cycle arrest. Cells that escape arrest show impaired metaphase plate formation and abnormal chromosomal segregation. Furthermore, the DNA2 inhibitor C5 mimics DNA2 knockout and synergistically kills cancer cells when combined with an ATR inhibitor. These findings provide mechanistic insights into how DNA2 supports replication of centromeric DNA and give further insights into new therapeutic strategies. Synopsis Whole genome screening uncovers an essential role of DNA2 helicase/nuclease, known to be involved in replication fork processing and double-strand break resection, in resolution of DNA secondary structures arising during replication of centromeric repeat DNA. Human DNA2 is specifically recruited to centromeric DNA regions of the genome. The nuclease and helicase activities of DNA2 are both essential for resolution of DNA structural obstacles to facilitate replication fork movement. Loss of DNA2-mediated clean-up mechanisms impairs centromeric replication and causes defects in CENP-A deposition, leading to ATR checkpoint activation and late-S/G2 cell cycle arrest. Combined application of the DNA2 and ATR inhibitors synergistically kills cancer cells and may offer new therapeutic strategies. Introduction The centromere is an epigenetically specified chromosomal locus in humans. It orchestrates the segregation of chromosomes during cell division and is defined by the presence of the histone H3 variant centromere protein A (CENP-A; Cleveland et al, 2003; Verdaasdonk & Bloom, 2011; McKinley & Cheeseman, 2016). The presence of the CENP-A nucleosome is sufficient to recruit the constitutive centromere-associated network and the mitotic kinetochore proteins, which are required for proper chromosome segregation (Barnhart et al, 2011; Guse et al, 2011; Mendiburo et al, 2011; Hori et al, 2013). Faithful replication of the genome, including the centromeric regions, is a challenging task; high accuracy and efficiency are needed to replicate approximately 3 billion human DNA base pairs during S-phase of the cell cycle, which lasts 6–8 h. Replication of the centromeres is even more challenging because of the presence of DNA secondary structure, and therefore likely requires specialized replication machinery. The centromeres of human chromosomes contain the largest tandem DNA family in the human genome. This family is called α-satellite DNA and has been extensively studied as a paradigm for understanding the genomic organization of tandem DNA (Schueler et al, 2001; Rudd et al, 2006). The fundamental α-satellite repeat unit consists of 171-base pair (bp) monomers, which are found in large, highly homologous arrays of up to several million base pairs at the centromeres of all human chromosomes. These tandem arrays are composed of either divergent monomers that have no detectable higher order structure or chromosome-specific higher order repeat units (HORs) characterized by distinct repeating linear arrangements of an integral set of 171-bp monomers (Rudd & Willard, 2004). This HOR structure correlates with centromere function (Schueler et al, 2001). However, the HOR structure also burdens the DNA replication machinery (Grady et al, 1992; Zhu et al, 1996; Aze et al, 2016), and requires a cellular mechanism that combines RNA/DNA helicase activity to resolve its secondary structure and structure-specific nuclease activity, such as that found in DNA2, to process DNA replication intermediates. Mechanistically, it is currently unknown how centromeric DNA is replicated. It is unlikely that during DNA replication general nucleases such as flap endonuclease 1 (FEN1) can process structured DNA sequences, such as those found in telomeres and centromeres (Henricksen et al, 2000; Tarantino et al, 2015). In contrast to FEN1, DNA2 has both helicase and endonuclease activities (Budd et al, 1995), which could work together to resolve these challenging DNA structures. The enzymatic activities of DNA2, which reside in a RecB-like nuclease domain, target single-stranded DNA (ssDNA; Bae et al, 1998), DNA flaps (Kao et al, 2004; Copeland & Longley, 2008; Stewart et al, 2010), and DNA secondary structures (Lee et al, 2013; Lin et al, 2013). The C-terminal superfamily 1 helicase domain of DNA2 can unwind kilobases of dsDNA from the 5′ end in vitro (Pinto et al, 2016). However, the biological functions of the helicase activity of DNA2 in cells are unknown. Genetic inactivation of either the helicase or nuclease activity of DNA2 in cells from a wide range of organisms, including yeast and humans, induces permanent cell cycle arrest (Budd & Campbell, 1997; Budd et al, 2000; Lee et al, 2000; Zheng et al, 2008; Duxin et al, 2009, 2012). In yeast, the cell death has been ascribed to the role of yeast Dna2 in processing DNA replication intermediates (Budd et al, 1995; Kang et al, 2000, 2010; Olmezer et al, 2016). In humans, DNA2-depleted cells undergo arrest during late-S/G2 phase of the cell cycle (Duxin et al, 2009, 2012). However, our understanding of the role of DNA2 in double-strand break (DSB) end resection cannot explain why DNA2 is required for viability in unperturbed cells (Zhu et al, 2008; Niu et al, 2009; Cejka et al, 2010; Chen et al, 2011), and indeed, the role of DNA2 during replication has yet to be defined. In the current study, we demonstrate that DNA2 predominantly binds to centromeric DNA regions. Single-molecule analysis of replicated DNA (SMARD) revealed that loss of DNA2 results in stalled replication of centromeres. These centromeric DNA replication defects led to activation of the DNA damage checkpoint kinase ataxia telangiectasia and Rad3 related (ATR), which contributes to cell cycle arrest in late-S/G2 phase. DNA2 nuclease- or helicase-deficient cells showed compromised loading of CENP-A onto chromatin and loss of intact centromeric DNA. In addition, cells that escaped the G2/M checkpoint showed inappropriate formation of the metaphase plate and chromosomal mis-segregation. Inhibition of both DNA2 and ATR had synergistic activity for killing breast, colorectal, and non-small-cell lung cancer cells. Collectively, these studies support a model wherein the concerted action of DNA2 helicase and nuclease activity is crucial to centromeric DNA replication. Results DNA2 binds preferentially to centromeric DNA and is required for centromeric DNA replication To explore the function of DNA2 in DNA replication, we first globally mapped the localization of DNA2 on chromatin. We used chromatin immunoprecipitation (ChIP) with a DNA2 antibody to pull down DNA2-associated chromatin in non-synchronized HCT-116 cells and then conducted whole-genome DNA sequencing. We found that DNA2 predominantly bound to the centromeric α-satellite regions (Figs 1A columns 1–3, and EV1A). This result was validated by using qPCR to compare the fold enrichment over input for the centromeric and non-centromeric regions (Fig 1B). The centromeric regions contained 58% of the DNA2-associated DNA, representing a 33.5-fold enrichment (Fig 1C). This finding supports our hypothesis that DNA2 is important in replication of centromeric DNA. In contrast to DNA2, when we used FEN1 ChIP DNA as a template, qPCR did not show any preferential recruitment of FEN1 to the centromeric DNA (Figs 1B and EV1B), suggesting these cellular nucleases have differing functions during DNA replication. Figure 1. DNA2 binds preferentially to centromeric DNA and is required for centromeric DNA replication DNA peaks were identified as described in Materials and Methods. Shown is an alignment of whole-genome sequencing results to the Hg38 genome. Column 1, Hg38 genome structure (UCSC genome browser); column 2, Hg38 centromere locations; column 3, DNA2 binding sites in HCT-116 cells identified from ChIP-seq; column 4, BrdU-negative DNA from DNA2-null cells; column 5, common peaks among DNA samples shown in lanes 3 and 4. The chromosome number is listed on the left of the plot. Fold enrichment of centromeric versus non-centromeric DNA over input as determined by qPCR of DNA2 and FEN1 ChIP samples. Shown by chromosome (Chr) are the means ± SDs of three biological repeats. See Table EV1 for primers used. Quantitative analysis of the DNA peaks and bases shown in panel (A). The most highly enriched repetitive centromeric/paracentromeric motif is shown. Letter size reflects its frequency at the position. P-value of 1.2e-88 derived by Fisher's exact test. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Controls for generating DNA libraries analyzed by whole-genome sequencing A, B. Western blot analysis to show efficiency of the DNA2 (A) and (B) Fen1 antibodies used in ChIP. Blots were probed for p84 to confirm specificity. C. Isolation of BrdU-negative DNA for whole-genome sequencing. DNA2Flox/−/− cells were treated with 4-OHT for 24 h, followed by incubation with 10 μM BrdU for 32 h. Cells were then fixed and sonicated, DNA was extracted, and BrdU-labeled DNA fragments were depleted. DNA samples from the indicated cells before (input) and after depletion of BrdU-labeled DNA were electrophoresed using a 2% agarose gel and stained with ethidium bromide. Source data are available online for this figure. Download figure Download PowerPoint Based on the preferential binding of DNA2 to centromeric DNA, we hypothesized that introducing a deficiency in DNA2 would retard centromeric DNA replication. To test this, we used HCT-116 cells engineered for Cre-mediated excision of DNA2 (DNA2Flox/−/−) in response to treatment with 4-hydroxytamoxifen (4-OHT, creating DNA2-null cells) and vehicle-treated cells (Karanja et al, 2014; Thangavel et al, 2015). We labeled newly synthesized DNA with the synthetic thymidine analog bromodeoxyuridine (BrdU) for 32 h, corresponding to at least one DNA2Flox/−/− cell cycle. The BrdU-incorporated nascent chromatin was then depleted using a BrdU antibody, leaving behind the BrdU-negative, under-replicated DNA. In 4-OHT-treated DNA2-null cells, approximately 10% of the genomic DNA was BrdU-negative, under-replicated DNA, but there was no detectable under-replicated DNA in the vehicle-treated cells (Fig EV1C). This suggests that DNA2 is required to complete DNA replication. To define the under-replicated DNA regions in DNA2-null cells, we conducted whole-genome DNA sequencing of the under-replicated DNA. After normalization to genomic input DNA from the same cells, 12.9% of the peaks from the under-replicated DNA aligned with the centromeric DNA regions (Fig 1A column 4, and C), representing an 8.5-fold enrichment (Fig 1C). In addition, among the peaks that overlapped between the DNA2 pull-down and under-replicated regions, two-thirds fell into the centromeric regions, representing a 48-fold enrichment (Fig 1A column 5, and C). We then computed the motifs derived from the common peaks. The number and percentage of peaks associated with each motif, the mean number of sites per peak, and the P-value for each motif are shown in Fig EV2. The most frequent motif was (TGGAA)n (Figs 1D and EV2), which is often found in centromeric DNA regions (Grady et al, 1992; Catasti et al, 1994; Kipling et al, 1995; Zhu et al, 1996; Barry et al, 1999). Thus, our data suggest that DNA2 preferentially localizes to the centromere and is required for centromeric DNA replication. Click here to expand this figure. Figure EV2. The top 10 motifs identified from ChIP-seq1Percentage of peaks containing the motif and their average occurrence in the peaks. 2The presence of motifs was ranked by their P-values that were generated by Fisher's exact test. Download figure Download PowerPoint Concerted action of the nuclease and helicase activities of DNA2 facilitates processing of intermediate structures during centromeric DNA replication During centromeric DNA replication, DNA secondary structures can arise from the single-stranded template DNA, which may block replication fork progression in cells. We analyzed the ability of human wild-type (WT) DNA2 (hDNA2), as well as D294A nuclease-deficient (ND) and K671E helicase-deficient (HD) hDNA2 mutant proteins (Masuda-Sasa et al, 2006; Lin et al, 2013) to cleave the DNA replication intermediates that formed at various regions of the genome. Figure 2A–C illustrates the DNA structures, and Fig 2D and E shows cleavage of 5′- and 3′-labeled substrates, respectively. We first confirmed the activity of WT hDNA2 and the hDNA2 mutant proteins using an established DNA flap DNA substrate (Lin et al, 2013). Consistent with previous reports (Masuda-Sasa et al, 2006; Lin et al, 2013), both WT and HD hDNA2, but not ND hDNA2, efficiently cleaved a typical flap ssDNA that lacked repetitive DNA sequences (Fig 2A, D, and E, lanes 2–11), sequentially cleaving ~ 10 nucleotides (nts) from the 5′ end. When incubated with a model DNA substrate mimicking the hairpin DNA structure generated by centromeric repetitive DNA, i.e., (TGGAA)6 (Fig 2B), WT hDNA2, but not the ND or HD mutants, effectively separated the DNA helix and cleaved the DNA structure, also ~ 10 nts from the 5′ end, in a sequential manner (Fig 2D and E, lanes 12–21). Because the highly enriched centromeric 171-nt α-satellite DNA that we found in our CHIP-seq experiments (Waye & Willard, 1986; Bloom, 2014) can form stable longer stem-loop structures (Aze et al, 2016), we designed a third DNA substrate that mimicked this structure (Fig 2C). We found that WT hDNA2, but not the ND or HD mutants, could effectively cleave the stem-loop structure (Fig 2D and E, lanes 22–31). Importantly, the combined helicase and nuclease activities of DNA2 were required to separate the 5′ complementary DNA, which then became ssDNA that was cleavable by DNA2 nuclease at cleavage sites clustered ~ 10 nts from the 5′ end of the single-stranded DNA (Fig 2C). We also designed additional DNA structures (Fig EV3), which contained one, two, or three stem/loop structures, and comprehensively mapped the cleavage sites based on the product sizes shown in the gel images. Our cleavage site mapping indicated that DNA2 nuclease activity only works on the ssDNA regions and requires its helicase activity, which is similar as previous demonstrations (Lin et al, 2013; Ronchi et al, 2013; Liu et al, 2016). This result supports the following model for resolution of multiple stem-loop structures during centromeric DNA replication: The DNA2 helicase activity first separates the stem to make a single ssDNA that is long enough (~ 10 nts) for nuclease cleavage; then, when the ssDNA at the junction is exposed, DNA2 cleaves the whole hairpin structure and removes the structured DNA at the replication fork. Figure 2. DNA2 helicase and nuclease activities are required for removal of in vitro centromeric DNA secondary structures A–C. Panel (A) shows flap DNA structure (lanes 2–11 in panels D and E). Panel (B) shows the (TGGAA)n motif structure (lanes 12–21 in panels D and E). Panel (C) shows α-satellite DNA structure (lanes 22–31 in panels D and E). Red arrows mark the cleavage sites. D, E. 5′-radiolabeled (panel D) or 3′-radiolabeled (panel E) non-centromeric DNA substrates (lanes 2–11) or centromeric substrates (lanes 12–31) were incubated with purified DNA2 for 5, 10, or 20 min. Representative images from at least three biological repeats are shown. The DNA2 cleavage signatures are shown in panels (A–C), along with a model that illustrates the resolution of DNA secondary structure, as predicted by the "RNAfold" software package. Resolving of the DNA substrates required different amounts of DNA2 protein: 0.5 ng for the DNA flap, 10 ng for (TGGAA)n, and 7.5 ng for the α-satellite stem-loop structure. Source data are available online for this figure. Source Data for Figure 2 [embj201796729-sup-0003-SDataFig2.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV3. DNA2 cleaves at the 5′ end of each complementary DNA where the loops are A. Schematic graphs show the predicted structures of each substrate from the RNAfold software package. The cleavage signatures of DNA2 on these substrates are shown by arrows. B, C. DNA2 activity on substrates with similar structures as those shown in (A). Three substrates were made from two (substrate 1), three (substrate 2), or four repeats (substrate 3) of the 17-nt CENP-B-boxes to mimic the α-satellite stem-loop structure. WT DNA2 (5 ng) was incubated with 1 pmole of (B) 5′- or (C) 3′-radiolabeled substrate for 5, 10, 20, 30, or 40 min. Each small red square marks a cleavage product. D–F. An intermediate DNA product (illustrated in D) was specifically designed to determine if the second loop of substrate 2, shown in panel (A), was preferentially cleaved by DNA2. WT DNA2 (5 ng) was incubated with the (E) 5′- or (F) 3′-radiolabeled substrates for the same amount of time as in panels (B and C) to assess the cleavage. Source data are available online for this figure. Download figure Download PowerPoint DNA2 is required for centromeric DNA replication fork initiation and progression To assess DNA replication activities at the centromeric region, we modified the SMARD assay that was previously established for studying telomere replication (Norio & Schildkraut, 2001; Drosopoulos et al, 2015). In the SMARD assay, the cells were labeled with iodo-deoxyuridine (IdU) for 4 h during a first labeling period, followed by labeling with chloro-deoxyuridine (CldU) for another 4 h in a second labeling period (Norio & Schildkraut, 2001; Demczuk et al, 2012; Drosopoulos et al, 2015). These 4-h labeling periods provided sufficient time to replicate the DNA regions analyzed, so that we could use the differential labeling, which represented different DNA replication stages, to analyze the initiation and progression of DNA replication forks across the genome (Norio & Schildkraut, 2001). Such a relatively long labeling period is particularly important for analyzing the replication of difficult-to-replicate DNA regions using the SMARD assay (Drosopoulos et al, 2015). Following this labeling protocol (Norio & Schildkraut, 2001; Demczuk et al, 2012; Drosopoulos et al, 2015), we then combed genomic DNA from these cells onto coverslips and used centromere-specific fluorescent DNA probes to identify centromeric DNA. Only DNA fibers that had IdU labeling and/or CldU labeling at non-centromeric or centromeric regions were analyzed to exclude including of DNA fibers from non-replicating cells. Presence of either red- or green-only tracks was classified as initiation events, while the length and distribution of the green segment within red-green tracks were evaluated to reflect the rate of replication progression. We found that the numbers of tracks with either red- or green-only and the lengths and distributions of green segments within red-green tracks at non-centromere regions in DNA2 mutant cells (null, ND, and HD) were similar to those in WT cells (Fig 3A and B), which agreed with a previous report (Thangavel et al, 2015). However, the numbers of tracks with only either red or green, and the lengths and distributions of green segments within red-green tracks at centromeric regions in DNA2 mutant cells were both remarkably reduced (Fig 3A and C). Twenty to thirty percent of DNA fibers displayed no detectable green or red tracks at centromeric regions in the DNA2 null, ND, or HD mutant cells as compared to only ~ 1% of fibers for WT cells (Fig 3A and C), indicating DNA replication initiation defects at centromere regions in mutant cells. With respect to red-green tracks, the median lengths of green segments at centromeric regions in DNA2 null, ND, and HD cells were 46.5, 40.3, and 54.6 μm, respectively, compared to a median length of 223.7 μm for centromeric green segments in WT cells (Fig 3C). Approximately 25% of these green tracks within centromeric DNA in DNA2-null and ND or HD mutant cells were 0–50 μm, and < 5% of the green tracks were longer than 210 μm. In contrast, < 5% of centromeric green tracks in WT cells were 0–50 μm, and approximately 70% of the green tracks were longer than 210 μm. These findings indicated defects in DNA replication initiation and replication fork progression at the centromeric regions in DNA2 mutant cells. Figure 3. DNA2-mutant cells show reduced replication initiation and slower centromeric replication fork progression assessed by single-molecule analysis of replicated DNA (SMARD) A. DNA2Flox/−/− cells that stably expressed DNA2-WT, DNA2-ND, and DNA2-HD were treated with 1 μM 4-OHT for 72 h to remove endogenous DNA2 and then cultured with IdU (40 μM) for 4 h followed by CldU (200 μM) for 4 h. Shown are representative images of combined centromere-specific DNA and surrounding non-centromeric DNA. Bottom panels show a focused view of the centromeric DNA regions. Scale bars, 10 μm. B, C. Quantification of the length and frequency of green tracks in the non-centromeric (B) and centromeric (C) DNA regions (out of three independent replicates, n ≥ 200 tracks were scored for each dataset). To exclude counting of compromised replication initiation (no any countable tracks) that could be caused by changes in cell cycle distribution, the fibers with centromeric DNA that had at least either red or green tracks were counted to ensure the cell was in S-phase. Median track lengths are indicated in parentheses. Download figure Download PowerPoint Both the nuclease and helicase enzymatic activities of DNA2 are required for centromeric DNA integrity Because our results suggested that DNA2 deficiency impairs centromeric DNA replication, we assessed centromeric DNA replication defects in DNA2-null cells with and without complementation with WT, ND, and HD DNA2. Using qPCR, we detected reduced abundance of intact centromeric DNA in DNA2-null cells as compared to vehicle-treated cells (Fig 4A). We also found a significant loss of intact centromeric DNA, which was rescued by WT DNA2, but not by the ND and HD DNA2 mutants (Fig 4A). This suggests that the DNA2 helicase or nuclease activity, or both, is critical for centromere maintenance. Figure 4. Conditional knockout of DNA2 or inactivation of its nuclease/helicase activity compromises centromere integrity A. DNA2Flox/−/− cells were transfected with DNA2-WT, DNA2-ND, and DNA2-HD constructs, selected with 250 μg/ml hygromycin B for 2 weeks, and then treated with 1 μM 4-OHT for 72 h
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