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

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