Hashimoto Y , Sadano K , Miyata N , Ito H , Tanaka H .

19K06617 MEXT ¦ Japan Society for the Promotion of Science (JSPS)

             cell proliferation
             CMG complex

	Synopsis Vertebrate DONSON is involved in replication fork stability as a component of the replisome complex. This study reveals that DONSON also functions as an initiator protein to promote CMG (Cdc45-MCM-GINS) helicase assembly through the interaction with GINS in the initiation of DNA replication. DONSON is required for CMG helicase assembly during DNA replication initiation. DONSON physically interacts with Cdc45, GINS, and Polε via its N-terminal disordered region. The DONSON-GINS interaction contributes to CMG helicase assembly. DONSON's chromatin association during replication initiation requires the pre-replicative complex, TopBP1, and kinase activities of S-CDK and DDK. S-CDK and DDK require DONSON function to trigger replication initiation.
	Figure 1. DONSON's chromatin association increases upon replication initiation and persists until the end of replication termination A. Comparison of primary structures of DONSON orthologs in Drosophila melanogaster, Homo sapiens, and Xenopus leavis. The percentages of sequence similarity and identity in each region are indicated outside and within parenthesis, respectively. NLS: Nuclear Localization Signal. aa: amino acids. B–E. (B, C, E) Characterization of DONSON's chromatin association. Sperm nuclei (5,000/μl) were incubated for the indicated times in 20 μl of interphase egg extract. Chromatin fractions were isolated and analyzed by immunoblotting. His-p27 (100 μg/ml, p27 (CDKi)), PHA-767491 (50 μM, Cdc7i), aphidicolin (10 μg/ml, aph), His-geminin (50 μg/ml, gem), NMS-873 (100 μM, p97i), and MLN-4924 (20 μM, Culi) were supplemented as indicated. (D) Fold increase of the chromatin-bound DONSON. Similar experiments with (B) and (C) were performed in triplicate and DONSON signal intensity was quantified by ImageJ. The mean ratio of the post-initiation (28 or 30 min) to the pre-initiation (14 or 15 min) DONSON intensities is shown in the bar graph. Error bar, mean ± standard deviation (SD). P-values were calculated using the unpaired t-test (two-tailed). ns, not significant.
	Figure EV1. The structure of DONSON and its deletion construct
	Figure 2. DONSON is required for replisome assembly during DNA replication initiation Immunodepletion from the interphase egg extract was performed using control IgG beads (mock) or anti-DONSON antibody beads (ΔDNS). Recombinant Xenopus DONSON (untagged) was added to the mock- and DONSON-depleted extracts at 0 μM (−), 0.06 μM (×1), 0.18 (×3), and 0.36 μM (×9) as indicated. The extracts (1 μl) were analyzed by immunoblotting, except lanes 9–11, where 0.4 (0.4), 0.2 (0.2), and 0.1 (0.1) μl were used. Since the anti-DONSON antibody was recognized by multiple bands, the presumable DONSON band was indicated by dotted lines in a vertically expanded blot. Asterisk (*), non-specific band. DONSON requirement for the replisome assembly. Sperm nuclei (5,000/μl) were incubated in 20 μl of the mock- and DONDON-depleted extracts as prepared in (A) for 60 min in the presence of 10 μg/ml aphidicolin. The extracts (0.5 μl) and isolated chromatin fractions were analyzed by immunoblotting. DONSON requirement for replisome assembly. Sperm nuclei were incubated for the indicated times and conditions in the egg extract and the chromatin fractions were analyzed by immunoblotting. Each of two types of anti-DONSON antibodies (α-DNS #1, α-DNS #2) was added at 14 μg/ml to suppress DONSON function.
	Figure 3. DONSON is required for DNA replication DONSON requirement for DNA replication. Sperm nuclei were incubated for 60 min in egg extract with no IgG (non), control IgG (α-control), or one of the two kinds of anti-DONSON antibodies (α-DNS #1, α-DNS #2) in a final concentration of 14 μg/ml. Cy3-dCTP was added to all of the reactions at 1 μΜ to visualize the DNA replication. After incubation, the nuclei were fixed, stained with Hoechst 33258, and observed under a fluorescent microscope. Representative images are shown above. Scale bar; 50 μm. The relative Cy3 intensity of > 100 nuclei was quantified using ImageJ and is shown in the box plot below. The boxes define the upper and lower quartiles; the whiskers define max to min values; the X-mark indicates each mean value; the central band indicates each median; the circles indicate outliers. P-values were calculated using the Mann–Whitney test. ns, not significant. DONSON requirement for DNA replication. Sperm nuclei (5,000/μl) were incubated for 60 min in 10 μl of the untreated (control), mock- (mock), and DONDON-depleted (ΔDNS) extracts with 2 μM Cy5-dUTP. Aphidicolin (aph) was added to the mock-depleted extract (2.5 μg/ml) as a negative control. Various versions of the recombinant Xenopus DONSON (1–6) were added to the DONSON-depleted extracts at about 0.06 μM. 1; untagged DONSON, 2 and 3; GST-DONSON, 4 and 5; GST-DONSON (residues 155–579) (ΔN154), 6; His-DONSON. In the reactions corresponding to lanes 3 and 5, 3C protease was added to separate the GST from DONSON. After incubation, the genomic DNA was isolated and subjected to 0.8% TAE agarose gel electrophoresis, followed by SYBR Gold staining. The Cy5 and SYBR Gold fluorescent signals show replicated DNA and total DNA, respectively. The same experiment was performed in triplicate; the relative Cy5 intensities to the control reaction were quantified using Image J, as shown in the bar graph below. Error bar, mean ± standard deviation (SD). P-values were calculated using the unpaired t-test (two-tailed). ns, not significant.
	Figure EV2. Requirement of Claspin for DNA replication in the DONSON-depleted extract
	Figure EV3. Involvement of checkpoint signaling in DNA replication in the DONSON-depleted extract
	Figure 4. DONSON is dispensable for replication restart and progression after fork stalling Salt-sensitivity of chromatin-bound DONSON. Sperm nuclei (5,000/μl) were incubated in 20 μl of egg extract under each of the conditions as indicated, and the chromatin fractions were isolated with normal buffer plus 0 (−), 0.1, or 0.2 M NaCl, and analyzed by immunoblotting. In addition, 0.5 μl of each extract (ext) was analyzed for comparison. Experimental procedure to investigate the requirement of DONSON in replication restart and progression after fork stalling. In this procedure, DNA replication was first initiated in the presence of aphidicolin, and the nuclear fractions containing the stalled replication forks were isolated and transferred to an aphidicolin-free extract in the presence of CDK inhibitors, in which replication restart and progression are allowed without a new origin firing. The chromatin fractions were isolated and analyzed by immunoblotting. The nuclei were observed under fluorescent microscopy as in Fig 3A. The relative Cy3 intensity of > 100 nuclei was quantified using ImageJ and is shown in the box plot. The boxes define the upper and lower quartiles; the whiskers define max to min values; the X-mark indicates each mean value; the central band indicates each median; the circles indicates outliers. α-C; control IgG. α-DNS; anti-DONSON antibody. Negative controls were prepared by incubating the sperm nuclei in the second extract without undergoing the first reaction (2nd ext/α-C, 2nd ext/α-DNS). P-values were calculated using the Mann–Whitney test. ns, not significant. (left) The isolated genomic DNA was analyzed in the same manner as in Fig 3B. (right) The same experiment was repeated six times. The relative Cy5 intensities to the control reaction (normal α-C) were quantified using Image J, as shown in the bar graph. Error bar, mean ± standard deviation (SD). P-values were calculated using the unpaired t-test (two-tailed). ns, not significant.
	Figure 5. Identification of GINS- and Polε-interacting motifs within DONSON Ability of DONSON deletions to support replisome assembly. The DONSON depletion experiment, as that in Fig 2B, was performed using 3 × FLAG-tagged versions of the full length (amino acid residues 1–579) or N-terminal/C-terminal deletions (amino acid residues 1–157, 133–579, 1–476, 1–369) of Xenopus (x) DONSON, whose final concentrations were about 0.06 μM. The signals derived from DONSON are indicated with dotted lines. His-p27 and His-geminin were used as negative controls. Asterisk (*), non-specific bands. Interaction of DONSON with the replication factors. 3 × FLAG-tagged full length (xF), N-terminal region residue 1–157 (xN), middle region residue 133–369 (xM), and C-terminal region residue 358–579 (xC) versions of Xenopus DONSON were added to an interphase egg extract at about 8.8 μM. After incubation for 15 min, the whole extract was subjected to a pull-down assay using FLAG-M2 beads, and the precipitated proteins were analyzed by immunoblotting (FLAG-IP). The extracts (2.5%) were analyzed for comparison (Input). (above) CLUSTALW alignment of the N-terminal amino acid sequences of DONSON in Homo sapiens (Hs) (residues 1–114), Gallus gallus (Gg) (residues 1–93) and Xenopus laevis (Xl) (residues 1–109). Identical amino acid (*), amino acid with identical nature (:), amino acid with similar nature (.). Two highly conserved amino acid stretches (PGYSP, RRNPF) are shadowed. (below) A three-dimensional structure of human DONSON predicted by the AlphaFold database. The amino acid numbers are indicated in the structure. Identification of GINS- and Polε-interacting motifs in the N-terminal region of DONSON. The FLAG-IP experiment, as in (B), was performed with 2 μM of 3 × FLAG-tagged versions of wild-type (xN-WT), P6A/Y8A (xN-2A), N67A/P68A/F69A (xN-3A), and P6A/Y8A/N67A/P68A/F69A (xN-5A) of Xenopus DONSON each with an N-terminal with 157 amino acids. The extracts (6.7%) were analyzed for comparison (Input). Interaction of human DONSON with Xenopus GINS and Polε in egg extract. The FLAG-IP experiment, as in (B), was performed with 1 μM of 3 × FLAG-tagged versions of wild-type (hF-WT), P6A/Y8A (hF-2A), N78A/P79A/F80A (hF-3A), P6A/Y8A/N78A/P79A/F80A (hF-5A), K489T (hF-KT) of full-length human DONSON each as well as 3 × FLAG-tagged wild-type Xenopus DONSON (xF-WT). The extracts (5%) were analyzed for comparison (Input).
	Figure EV4. Interaction of DONSON with GINS is important for DNA replication initiation
	Figure 6. Interaction of DONSON with GINS is required for efficient DNA replication Requirement of GINS- and Polε-interacting motifs within DONSON for replisome assembly. This DONSON depletion experiment, as that in Fig 2B, was performed using the same recombinant proteins as in Fig 5E. Human proteins were added at 0.3 μM each and Xenopus protein at 0.06 μM. His-p27 (p27) and His-geminin (gem) were used as negative controls. Requirement of GINS- and Polε-interacting motifs within DONSON for replication activity. The same experiment procedure was used as in Fig 3A for the mock- and DONSON-depleted extracts with the same recombinant proteins as in Fig 5E. Xenopus proteins were added at 0.06 μM each while human proteins were added at 0.3 μM. The relative Cy3 intensity of > 100 nuclei was quantified using ImageJ and is shown in the box plot. The boxes define the upper and lower quartiles; the whiskers define max to min values; the X-mark indicates each mean value; the central band indicates each median; the circles indicates outliers. P-values were calculated using the Mann–Whitney test. ns, not significant.
	Figure 7. CDK and DDK cannot accomplish their functions in the replisome assembly without DONSON A, B. Requirement of GINS, Cdc45, and TopBP1 for the DONSON chromatin association. Sperm nuclei (5,000/μl) were incubated in 20 μl of mock-, GINS-, Cdc45- or TopBP1- depleted egg extract (ΔGINS, ΔCdc45, ΔTopBP1) for the indicated times and conditions, and the chromatin fractions were isolated and analyzed by immunoblotting. The extracts (0.5 μl) were also analyzed. Replication proteins were categorized into three groups (Group-1, −2, −3) based on their dependency on GINS, Cdc45, and TopBP1. C. (above) Requirement of DONSON for CDK and DDK function in the replisome assembly. Sperm nuclei (5,000/μl) were incubated for 50 min in 120 μl of egg extract with control IgG (α-cont) or anti-DONSON antibody #1 (α-DNS). Each part of this first reaction (1st) was divided equally into six parts, and the nuclear fractions were isolated and transferred to 20 μl of flesh extract with control IgG (α-cont) or anti-DONSON antibody #1 (α-DNS) in the presence or absence of CDK inhibitors (Ci: 100 μM roscovitine, 100 μg/ml His-p27) and Cdc7 inhibitor (7i: 50 μM PHA-767491). Aphidicolin was added to the first and second extracts at 10 μg/ml to suppress replication termination. After incubation for 40 min in the second reaction (2nd), the chromatin fractions were isolated and analyzed by immunoblotting. In addition, the extracts (0.5 μl) were analyzed (ext). (below) It is summarized whether or not replisome assembly was allowed under each condition, where DONSON, CDK, and Cdc7 were either functional (+) or non-functional (−) in the 1st and 2nd incubations. D. Model of DONSON function in the replisome assembly based on our observations.
	Figure EV5. Effect of phosphatase inhibitors on DNA replication

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

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
Attali, Structural Mechanisms for Replicating DNA in Eukaryotes. 2021, Pubmed
Bandura, humpty dumpty is required for developmental DNA amplification and cell proliferation in Drosophila. 2005, Pubmed
Bellelli, Spotlight on the Replisome: Aetiology of DNA Replication-Associated Genetic Diseases. 2021, Pubmed
Bonnon, PGY repeats and N-glycans govern the trafficking of paranodin and its selective association with contactin and neurofascin-155. 2007, Pubmed
Boos, Identification of a heteromeric complex that promotes DNA replication origin firing in human cells. 2013, Pubmed
Burgers, Eukaryotic DNA Replication Fork. 2017, Pubmed
Calvi, Cell cycle control of chorion gene amplification. 1998, Pubmed
Cho, CDC7 kinase phosphorylates serine residues adjacent to acidic amino acids in the minichromosome maintenance 2 protein. 2006, Pubmed
Costa, The Initiation of Eukaryotic DNA Replication. 2022, Pubmed
de Beer, Molecular mechanism of NPF recognition by EH domains. 2000, Pubmed
Deegan, Phosphopeptide binding by Sld3 links Dbf4-dependent kinase to MCM replicative helicase activation. 2016, Pubmed
de Munnik, Meier-Gorlin syndrome. 2015, Pubmed
Dewar, Mechanisms of DNA replication termination. 2017, Pubmed , Xenbase
Douglas, Recruitment of Mcm10 to Sites of Replication Initiation Requires Direct Binding to the Minichromosome Maintenance (MCM) Complex. 2016, Pubmed
Douglas, The mechanism of eukaryotic CMG helicase activation. 2018, Pubmed
Evrony, Integrated genome and transcriptome sequencing identifies a noncoding mutation in the genome replication factor DONSON as the cause of microcephaly-micromelia syndrome. 2017, 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
Gillespie, DDK: The Outsourced Kinase of Chromosome Maintenance. 2022, Pubmed
Hashimoto, Xenopus Cut5 is essential for a CDK-dependent process in the initiation of DNA replication. 2003, Pubmed , Xenbase
Hashimoto, Mitotic entry drives replisome disassembly at stalled replication forks. 2018, Pubmed , Xenbase
Hashimoto, Ongoing replication forks delay the nuclear envelope breakdown upon mitotic entry. 2021, Pubmed , Xenbase
Hashimoto, Mre11 exonuclease activity promotes irreversible mitotic progression under replication stress. 2022, Pubmed , Xenbase
Hashimoto, Rad51 protects nascent DNA from Mre11-dependent degradation and promotes continuous DNA synthesis. 2010, Pubmed , Xenbase
Hashimoto, RAD51- and MRE11-dependent reassembly of uncoupled CMG helicase complex at collapsed replication forks. 2011, Pubmed , Xenbase
Heller, Eukaryotic origin-dependent DNA replication in vitro reveals sequential action of DDK and S-CDK kinases. 2011, Pubmed
Huang, The DNA translocase FANCM/MHF promotes replication traverse of DNA interstrand crosslinks. 2013, Pubmed
Huang, Remodeling of Interstrand Crosslink Proximal Replisomes Is Dependent on ATR, FANCM, and FANCD2. 2019, Pubmed
Jares, Xenopus cdc7 function is dependent on licensing but not on XORC, XCdc6, or CDK activity and is required for XCdc45 loading. 2000, Pubmed , Xenbase
Kamada, Structure of the human GINS complex and its assembly and functional interface in replication initiation. 2007, Pubmed
Kamimura, Sld2, which interacts with Dpb11 in Saccharomyces cerevisiae, is required for chromosomal DNA replication. 1998, Pubmed
Knapp, Linked-read genome sequencing identifies biallelic pathogenic variants in DONSON as a novel cause of Meier-Gorlin syndrome. 2020, Pubmed
Kubota, Determination of initiation of DNA replication before and after nuclear formation in Xenopus egg cell free extracts. 1993, Pubmed , Xenbase
Kubota, A novel ring-like complex of Xenopus proteins essential for the initiation of DNA replication. 2003, 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, Treslin collaborates with TopBP1 in triggering the initiation of DNA replication. 2010, Pubmed , Xenbase
Kumagai, Direct regulation of Treslin by cyclin-dependent kinase is essential for the onset of DNA replication. 2011, Pubmed , Xenbase
Lesly, Rapid DNA Synthesis During Early Drosophila Embryogenesis Is Sensitive to Maternal Humpty Dumpty Protein Function. 2017, Pubmed
Li, The Eukaryotic CMG Helicase at the Replication Fork: Emerging Architecture Reveals an Unexpected Mechanism. 2018, Pubmed , Xenbase
Lin, Replication initiation: Implications in genome integrity. 2021, Pubmed
Mann, Defective sister-chromatid cohesion, aneuploidy and cancer predisposition in a mouse model of type II Rothmund-Thomson syndrome. 2005, Pubmed
Maric, Cdc48 and a ubiquitin ligase drive disassembly of the CMG helicase at the end of DNA replication. 2014, Pubmed
Masai, Phosphorylation of MCM4 by Cdc7 kinase facilitates its interaction with Cdc45 on the chromatin. 2006, 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
McGarry, Geminin, an inhibitor of DNA replication, is degraded during mitosis. 1998, Pubmed , Xenbase
Mimura, Xenopus Cdc45-dependent loading of DNA polymerase alpha onto chromatin under the control of S-phase Cdk. 1998, Pubmed , Xenbase
Moiseeva, Regulation of the initiation of DNA replication in human cells. 2018, Pubmed , Xenbase
Montagnoli, A Cdc7 kinase inhibitor restricts initiation of DNA replication and has antitumor activity. 2008, Pubmed
Moreno, Mechanisms of eukaryotic replisome disassembly. 2020, Pubmed , Xenbase
Moreno, Polyubiquitylation drives replisome disassembly at the termination of DNA replication. 2014, Pubmed , Xenbase
Morgan, Eps15 homology domain-NPF motif interactions regulate clathrin coat assembly during synaptic vesicle recycling. 2003, Pubmed
Muramatsu, CDK-dependent complex formation between replication proteins Dpb11, Sld2, Pol (epsilon}, and GINS in budding yeast. 2010, Pubmed
Parker, Mechanisms and regulation of DNA replication initiation in eukaryotes. 2017, Pubmed
Ramer, Dbf4 and Cdc7 proteins promote DNA replication through interactions with distinct Mcm2-7 protein subunits. 2013, Pubmed
Randell, Mec1 is one of multiple kinases that prime the Mcm2-7 helicase for phosphorylation by Cdc7. 2010, Pubmed
Reynolds, Mutations in DONSON disrupt replication fork stability and cause microcephalic dwarfism. 2017, 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
Schulz, Microcephaly, short stature, and limb abnormality disorder due to novel autosomal biallelic DONSON mutations in two German siblings. 2018, Pubmed
Semlow, Mechanisms of Vertebrate DNA Interstrand Cross-Link Repair. 2021, Pubmed
Sheu, Cdc7-Dbf4 phosphorylates MCM proteins via a docking site-mediated mechanism to promote S phase progression. 2006, Pubmed
Sheu, The Dbf4-Cdc7 kinase promotes S phase by alleviating an inhibitory activity in Mcm4. 2010, Pubmed
Tada, Repression of origin assembly in metaphase depends on inhibition of RLF-B/Cdt1 by geminin. 2001, Pubmed , Xenbase
Tak, A CDK-catalysed regulatory phosphorylation for formation of the DNA replication complex Sld2-Dpb11. 2006, Pubmed
Takahashi, Cdc7-Drf1 is a developmentally regulated protein kinase required for the initiation of vertebrate DNA replication. 2005, Pubmed , Xenbase
Takayama, GINS, a novel multiprotein complex required for chromosomal DNA replication in budding yeast. 2003, Pubmed
Tanaka, Origin association of Sld3, Sld7, and Cdc45 proteins is a key step for determination of origin-firing timing. 2011, Pubmed
Tanaka, Efficient initiation of DNA replication in eukaryotes requires Dpb11/TopBP1-GINS interaction. 2013, Pubmed , Xenbase
Tanaka, Helicase activation and establishment of replication forks at chromosomal origins of replication. 2013, Pubmed
Tanaka, CDK-dependent phosphorylation of Sld2 and Sld3 initiates DNA replication in budding yeast. 2007, Pubmed
Tsuji, Essential role of phosphorylation of MCM2 by Cdc7/Dbf4 in the initiation of DNA replication in mammalian cells. 2006, Pubmed
van Deursen, Mcm10 associates with the loaded DNA helicase at replication origins and defines a novel step in its activation. 2012, Pubmed
Van Hatten, The Xenopus Xmus101 protein is required for the recruitment of Cdc45 to origins of DNA replication. 2002, Pubmed , Xenbase
Volpi, The role of DDK and Treslin-MTBP in coordinating replication licensing and pre-initiation complex formation. 2021, Pubmed , Xenbase
Walter, Evidence for sequential action of cdc7 and cdk2 protein kinases during initiation of DNA replication in Xenopus egg extracts. 2000, Pubmed , Xenbase
Xu, MCM10 mediates RECQ4 association with MCM2-7 helicase complex during DNA replication. 2009, Pubmed , Xenbase
Xu, Molecular Mechanisms of the RECQ4 Pathogenic Mutations. 2021, Pubmed
Yeeles, Regulated eukaryotic DNA replication origin firing with purified proteins. 2015, Pubmed
Zegerman, Evolutionary conservation of the CDK targets in eukaryotic DNA replication initiation. 2015, Pubmed
Zegerman, Phosphorylation of Sld2 and Sld3 by cyclin-dependent kinases promotes DNA replication in budding yeast. 2007, Pubmed
Zhang, DNA interstrand cross-link repair requires replication-fork convergence. 2015, Pubmed , Xenbase
Zhang, DONSON and FANCM associate with different replisomes distinguished by replication timing and chromatin domain. 2020, Pubmed
Zhang, Replication of the Mammalian Genome by Replisomes Specific for Euchromatin and Heterochromatin. 2021, Pubmed