Matsuno K , Kumano M , Kubota Y , Hashimoto Y , Takisawa H .

             DNA replication

	FIG. 1. Sequence comparison of Sld2, Drc1, and RecQ4. (A) Schematic structures of Sld2/Drc1 of Saccharomyces cerevisiae (Sc), Schizosaccharomyces pombe (Sp), and Xenopus laevis (Xl) RecQ4. Potential CDK phosphorylation motifs (S/T-P, S/T-P-K/R, and S/T-P-X-K/R) are indicated by open (one motif) and solid (two motifs) inverted triangles. The black region represents the RecQ helicase domain (amino acids 744 to 1086). Bold lines represent N-terminal fragments of Xenopus RecQ4 used in this study. Regions 1, 2, and 3 show the following identity and similarity (% identity/% similarity): Xl RecQ4/Sc Sld2, 30/55, 19/31, and 19/35; Xl RecQ4/Sp Drc1, 35/52, 16/28, and 23/34; and Sc Sld2/Sp Drc1, 29/55, 26/40, and 20/32, respectively. (B) Alignment of N-terminal amino acid sequences of Sc Sld2/Drc1, Sp Drc1, and Xl RecQ4. Identical amino acids are indicated just below the sequence of Sp Drc1, and conserved regions are shaded. Boxed regions show relatively high sequence similarities.
	FIG. 2. Chromatin binding of RecQ4 in interphase extracts of Xenopus eggs. (A) Specificity of anti-RecQ4 antibody. Interphase egg extract was resolved by SDS-PAGE and immunoblotted with anti-RecQ4 antibody. (B) Time course of chromatin binding of RecQ4 and other replication proteins under various conditions. Xenopus sperm chromatin was incubated in egg extracts at 23°C in the absence (Control) or the presence of 15 μg/ml green fluorescent protein-geminin (+geminin), 50 μg/ml GST-p21 plus 200 μM roscovitine (+p21/ros.), or 40 μg/ml aphidicolin (+Aphi) for the indicated times. The chromatin fractions isolated by centrifugation and the egg extract (1 μl) were resolved by SDS-PAGE and immunoblotted with antibodies indicated in the figure. Negative control of chromatin fractions was obtained by incubating the extracts in the absence of sperm chromatin (−sperm).
	FIG. 3. Effect of RecQ4 depletion from the egg extract on DNA replication and chromatin binding of DNA polymerase α. (A) Depletion of RecQ4 from the extracts. The egg extracts were treated with preimmune or anti-RecQ4 antibodies conjugated to protein A beads. Untreated egg extract and mock- and RecQ4-depleted extracts (1 μl) were resolved by SDS-PAGE and immunoblotted with the antibodies indicated in the figure. (B) Immunofluorescent detection of nuclear RecQ4. Xenopus sperm chromatin was incubated in mock- and RecQ4-depleted extracts for 40 min at 23°C. The samples were treated with 0.1% NP-40 and then fixed with 3.7% formaldehyde in EB. Nuclear localization of RecQ4 was visualized with rabbit anti-RecQ4 antibody followed by Alexa 488-labeled anti-rabbit immunoglobulin G. DNA replications were monitored as the incorporation of Cy3-dCTP into DNA, and DNA was visualized with Hoechst 33258 dye. Fluorescence images were captured with the OpenLab imaging program (Improvision). (C) Chromatin binding of Cut5 in the absence of RecQ4. Xenopus sperm chromatin was incubated in mock- or RecQ4-depleted extracts for 40 min at 23°C. Isolated chromatin fractions were resolved by SDS-PAGE and immunoblotted with the antibodies indicated. (D) Chromatin binding of RecQ4 in the absence of Cut5. Xenopus sperm chromatin was incubated in mock- or Cut5-depleted extracts with (+) or without (−) recombinant Cut5 for 40 min at 23°C.
	FIG. 4. Effect of caffeine on DNA replication in mock- or RecQ4-depleted extracts. (A) Xenopus sperm chromatin was incubated in mock- (ΔMock) or RecQ4-depleted (ΔRecQ4) extracts with (+) or without (−) 5 mM caffeine for indicated times at 23°C. DNA replication activity was monitored as the incorporation of [α-32P]dCTP into DNA. At the times indicated, DNA was subjected to agarose gel electrophoresis and then autoradiographed. [α-32P]dCTP incorporation was carried out as described previously (24) except that the autoradiography was quantified by Image Gauge software (Fuji Film). (B) The amount of 32P incorporated into DNA in panel A was quantified, taking the highest value of the mock-depleted extracts as 100%.
	FIG. 5. Rescue of DNA replication activity and nuclear localization of DNA polymerase α by recombinant RecQ4 in RecQ4-depleted extracts. (A) Sperm chromatin was incubated in mock (ΔMock)- or RecQ4 (ΔRecQ4)-depleted extracts in the absence (−) or presence of full-length RecQ4 (+low-full, 2 μg/ml), N2-RecQ4 (+low-N2, 0.8 μg/ml; +high-N2, 16 μg/ml), or N1-RecQ4 (+high-N1, 9 μg/ml) at 23°C for 40 min. The samples were treated for 2 min with 0.1% Triton X-100 in EB containing an additional 100 mM NaCl and then fixed with 3.7% formaldehyde. Nuclear localization of DNA polymerase α was visualized with rabbit anti-polymerase α antibody followed by Alexa 488-labeled anti-rabbit immunoglobulin G. DNA replication was monitored as the incorporation of Cy3-dCTP into DNA, and DNA was visualized with Hoechst 33258 dye. (B) The fluorescent intensity of each nucleus was quantified using NIH Image software, and average intensities of over 40 nuclei were normalized by the intensities of the nuclei from mock-depleted extract.
	FIG. 6. Rescue of DNA replication activity of RecQ4-depleted extracts by N-terminal fragments of RecQ4. (A) SDS-polyacrylamide gel showing recombinant N-terminal fragments of Xenopus RecQ4. N1- and N2-RecQ4 were expressed in Sf9 cells using a baculovirus expression system. The proteins purified through Ni-nitrilotriacetic acid resin were resolved by SDS-PAGE and visualized with Coomassie blue staining. (B and C) Rescue of DNA replication by N-terminal fragments of RecQ4. Sperm chromatin was incubated in mock- (ΔMock) or RecQ4-depleted (ΔRecQ4) extracts in the absence (−) or presence of N2-RecQ4 (+N2, 16 μg/ml) or N1-RecQ4 (+N1, 9 μg/ml) at 23°C. DNA replication activity was monitored as the incorporation of [α-32P]dCTP into DNA. At the indicated times, DNA was isolated, electrophoresed in an agarose gel, and autoradiographed. The bar graph (C) shows the amount of 32P incorporated into DNA, taking the highest value of the mock-depleted extracts as 100%. (D) Recovery of chromatin binding of DNA polymerase α by N-terminal fragments of RecQ4 in RecQ4-depleted extracts. Xenopus sperm chromatin was incubated in mock (ΔMock)- or RecQ4 (ΔRecQ4)-depleted extracts in the absence (−) or presence of N2-RecQ4 (+N2, 16 μg/ml) for the indicated times at 23°C. Isolated chromatin fractions were resolved by SDS-PAGE and immunoblotted with the indicated antibodies.
	FIG. 7. Inhibition of DNA replication and chromatin binding of DNA polymerase α by anti-RecQ4 antibody. (A) Xenopus sperm chromatin was incubated in egg extract containing 72.5 μg/ml of preimmune antibody (αControl) or anti-RecQ4 (αRecQ4) antibody in the absence (−) or presence of N-terminal 727-amino-acid fragments of RecQ4 (+N3, 82.7 μg/ml). DNA replication activity was monitored as the incorporation of [α-32P]dCTP into DNA. At the indicated times, DNA was isolated, electrophoresed in an agarose gel, and autoradiographed. The amount of 32P incorporated into DNA was quantified, taking the highest value of the control extracts as 100%. (B) Effect of anti-RecQ4 antibody on the chromatin binding of various replication proteins. Xenopus sperm chromatin was incubated in egg extract as in panel A, and the chromatin fractions were collected at the indicated times. Chromatin fractions and 1 μl of egg extract were resolved by SDS-PAGE and immunoblotted with the indicated antibodies. Negative control of chromatin fractions was obtained by incubating the extracts in the absence of sperm chromatin (−sperm).
	FIG. 8. Physical interaction between RecQ4 and Cut5. (A) Coimmunoprecipitation of RecQ4 and Cut5. The egg extracts (80 μl) were incubated with preimmune control immunoglobulin G (Control), anti-RecQ4 (RecQ4), or anti-Cut5 (Cut5) antibody-conjugated protein A beads. The untreated 1 μl of egg extract, flowthrough extracts (FT), and the proteins bound to the beads (IP) were resolved by SDS-PAGE and blotted onto nitrocellulose membrane for immunostaining. (B) Physical interaction of RecQ4 and Cut5 in vitro. Recombinant N1- and N2-RecQ4 fragments were incubated with anti-Cut5 antibody (−) or the antibody prebound with Cut5 (+) conjugated to protein A beads, and proteins bound to the antibody were pulled down with the beads. Input fragments (Input) and immunoprecipitated fragments (IP) were resolved by SDS-PAGE and blotted onto a nitrocellulose membrane for immunostaining. Arrowheads indicate the position of each fragment. *, degradation product of Cut5. (C) Effect of phosphorylation of RecQ4 on the interaction with Cut5. Recombinant N2-RecQ4 fragment was treated with 5.6 μg/ml protein phosphatase 1 with (+) or without (−) phosphatase inhibitor (1 μM okadaic acid). For rephosphorylation, the sample treated with the phosphatase was further incubated with 17 μg/ml cyclin A/Cdk2 in the presence of 1 μM okadaic acid to prevent the dephosphorylation. The samples were then incubated with Cut5 prebound to anti-Cut5 antibody. Both input (Input) and immunoprecipitated N2 fragments (IP) were resolved by SDS-PAGE and blotted onto a nitrocellulose membrane for immunostaining. (D) Physical interaction of unphosphorylated RecQ4 and Cut5 in vitro. The experiment was carried out as described for panel B except that the N2 fragment expressed in E. coli was used instead of the protein expressed in Sf9 cells.

Bachrati, RecQ helicases: suppressors of tumorigenesis and premature aging. 2003, Pubmed

Bachrati, RecQ helicases: suppressors of tumorigenesis and premature aging. 2003, Pubmed
Balraj, An unusual mutation in RECQ4 gene leading to Rothmund-Thomson syndrome. 2002, Pubmed
Bell, DNA replication in eukaryotic cells. 2002, Pubmed
Bjergbaek, Mechanistically distinct roles for Sgs1p in checkpoint activation and replication fork maintenance. 2005, Pubmed
Blow, Replication licensing--defining the proliferative state? 2002, Pubmed
Cobb, DNA polymerase stabilization at stalled replication forks requires Mec1 and the RecQ helicase Sgs1. 2003, Pubmed
Furuichi, Premature aging and predisposition to cancers caused by mutations in RecQ family helicases. 2001, Pubmed
Hashimoto, Xenopus Cut5 is essential for a CDK-dependent process in the initiation of DNA replication. 2003, Pubmed , Xenbase
Hickson, RecQ helicases: caretakers of the genome. 2003, Pubmed
Hoki, Growth retardation and skin abnormalities of the Recql4-deficient mouse. 2003, Pubmed
Ichikawa, [Preparation of the gene targeted knockout mice for human premature aging diseases, Werner syndrome, and Rothmund-Thomson syndrome caused by the mutation of DNA helicases]. 2002, Pubmed
Kamimura, Sld3, which interacts with Cdc45 (Sld4), functions for chromosomal DNA replication in Saccharomyces cerevisiae. 2001, Pubmed
Kamimura, Sld2, which interacts with Dpb11 in Saccharomyces cerevisiae, is required for chromosomal DNA replication. 1998, Pubmed
Kanemaki, Functional proteomic identification of DNA replication proteins by induced proteolysis in vivo. 2003, Pubmed
Kearsey, Enigmatic variations: divergent modes of regulating eukaryotic DNA replication. 2003, Pubmed
Kitao, Mutations in RECQL4 cause a subset of cases of Rothmund-Thomson syndrome. 1999, 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
Kubota, Identification of the yeast MCM3-related protein as a component of Xenopus DNA replication licensing factor. 1995, Pubmed , Xenbase
Lindor, Rothmund-Thomson syndrome due to RECQ4 helicase mutations: report and clinical and molecular comparisons with Bloom syndrome and Werner syndrome. 2000, Pubmed
Macris, Biochemical characterization of the RECQ4 protein, mutated in Rothmund-Thomson syndrome. 2006, Pubmed
Marheineke, Control of replication origin density and firing time in Xenopus egg extracts: role of a caffeine-sensitive, ATR-dependent checkpoint. 2004, Pubmed , Xenbase
Masumoto, S-Cdk-dependent phosphorylation of Sld2 essential for chromosomal DNA replication in budding yeast. 2002, Pubmed
Masumoto, Dpb11 controls the association between DNA polymerases alpha and epsilon and the autonomously replicating sequence region of budding yeast. 2000, Pubmed
Méndez, Perpetuating the double helix: molecular machines at eukaryotic DNA replication origins. 2003, Pubmed , Xenbase
Mimura, Central role for cdc45 in establishing an initiation complex of DNA replication in Xenopus egg extracts. 2000, Pubmed , Xenbase
Mimura, Xenopus Cdc45-dependent loading of DNA polymerase alpha onto chromatin under the control of S-phase Cdk. 1998, Pubmed , Xenbase
Nakajima, SpSld3 is required for loading and maintenance of SpCdc45 on chromatin in DNA replication in fission yeast. 2002, Pubmed
Nishitani, Control of DNA replication licensing in a cell cycle. 2002, Pubmed , Xenbase
Noguchi, CDK phosphorylation of Drc1 regulates DNA replication in fission yeast. 2002, Pubmed
Sangrithi, Initiation of DNA replication requires the RECQL4 protein mutated in Rothmund-Thomson syndrome. 2005, Pubmed , Xenbase
Tada, Repression of origin assembly in metaphase depends on inhibition of RLF-B/Cdt1 by geminin. 2001, Pubmed , Xenbase
Takayama, GINS, a novel multiprotein complex required for chromosomal DNA replication in budding yeast. 2003, Pubmed
Van Hatten, The Xenopus Xmus101 protein is required for the recruitment of Cdc45 to origins of DNA replication. 2002, Pubmed , Xenbase
Walter, Initiation of eukaryotic DNA replication: origin unwinding and sequential chromatin association of Cdc45, RPA, and DNA polymerase alpha. 2000, Pubmed , Xenbase
Wang, DRC1, DNA replication and checkpoint protein 1, functions with DPB11 to control DNA replication and the S-phase checkpoint in Saccharomyces cerevisiae. 1999, Pubmed
Wohlschlegel, Inhibition of eukaryotic DNA replication by geminin binding to Cdt1. 2000, Pubmed , Xenbase
Yoshida, Intrinsic nuclear import activity of geminin is essential to prevent re-initiation of DNA replication in Xenopus eggs. 2005, Pubmed , Xenbase