The effects of the nucleotide compositions of the 3′-tail on displacement of partial heteroduplex substrates by Mcm4/6/7

<p><b>Copyright information:</b></p><p>Taken from "DNA binding and helicase actions of mouse MCM4/6/7 helicase"</p><p>Nucleic Acids Research 2005;33(9):3033-3047.</p><p>Published online 25 May 2005</p><p>PMCID:PMC1140370.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> () DNA helicase assays were performed with 3′-tailed partial heteroduplex helicase substrates (on a single-stranded circular DNA; 4 fmol) carrying various nucleotide sequences in the 3′-tail as shown. The asterisks represent the P-labeled 5′ ends of the annealed oligonucleotides. () DNA helicase assays were performed with similar sets of partial heteroduplex helicase substrates carrying the 3′-tails as shown. () Quantification of displaced oligonucleotides in (B). Lanes 1–4 contain 0, 25, 50 and 100 ng of the Mcm4/6/7 complex, respectively. B, boiled substrate

DNA binding and helicase actions of Mcm4/6/7 on 5′- and 3′-extension substrates

<p><b>Copyright information:</b></p><p>Taken from "DNA binding and helicase actions of mouse MCM4/6/7 helicase"</p><p>Nucleic Acids Research 2005;33(9):3033-3047.</p><p>Published online 25 May 2005</p><p>PMCID:PMC1140370.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> DNA-binding () and helicase () activities of Mcm4/6/7 were examined on various 3′-extension substrates (4 fmol) as shown. () Quantification of the displaced substrates in (B). () DNA helicase assays on 5′-extension, 3′-extension and Y-forked substrates. Lanes 1–4 are reactions with 0, 25, 50 and 100 ng of the Mcm4/6/7 complex, respectively. Schematic drawings of the substrates used in the assays are also shown. The star marks indicate P-labeled 5′-termini

Characterization of conserved arginine residues on Cdt1 that affect licensing activity and interaction with Geminin or Mcm complex

<p>All organisms ensure once and only once replication during S phase through a process called replication licensing. Cdt1 is a key component and crucial loading factor of Mcm complex, which is a central component for the eukaryotic replicative helicase. In higher eukaryotes, timely inhibition of Cdt1 by Geminin is essential to prevent rereplication. Here, we address the mechanism of DNA licensing using purified Cdt1, Mcm and Geminin proteins in combination with replication in <i>Xenopus</i> egg extracts. We mutagenized the 223th arginine of mouse Cdt1 (mCdt1) to cysteine or serine (R-S or R-C, respectively) and 342nd and 346th arginines constituting an arginine finger-like structure to alanine (RR-AA). The RR-AA mutant of Cdt1 could not only rescue the DNA replication activity in Cdt1-depleted extracts but also its specific activity for DNA replication and licensing was significantly increased compared to the wild-type protein. In contrast, the R223 mutants were partially defective in rescue of DNA replication and licensing. Biochemical analyses of these mutant Cdt1 proteins indicated that the RR-AA mutation disabled its functional interaction with Geminin, while R223 mutations resulted in ablation in interaction with the Mcm2∼7 complex. Intriguingly, the R223 mutants are more susceptible to the phosphorylation-induced inactivation or chromatin dissociation. Our results show that conserved arginine residues play critical roles in interaction with Geminin and Mcm that are crucial for proper conformation of the complexes and its licensing activity.</p

The Mini-Chromosome Maintenance (Mcm) Complexes Interact with DNA Polymerase α-Primase and Stimulate Its Ability to Synthesize RNA Primers

<div><p>The Mini-chromosome maintenance (Mcm) proteins are essential as central components for the DNA unwinding machinery during eukaryotic DNA replication. DNA primase activity is required at the DNA replication fork to synthesize short RNA primers for DNA chain elongation on the lagging strand. Although direct physical and functional interactions between helicase and primase have been known in many prokaryotic and viral systems, potential interactions between helicase and primase have not been explored in eukaryotes. Using purified Mcm and DNA primase complexes, a direct physical interaction is detected in pull-down assays between the Mcm2∼7 complex and the hetero-dimeric DNA primase composed of the p48 and p58 subunits. The Mcm4/6/7 complex co-sediments with the primase and the DNA polymerase α-primase complex in glycerol gradient centrifugation and forms a Mcm4/6/7-primase-DNA ternary complex in gel-shift assays. Both the Mcm4/6/7 and Mcm2∼7 complexes stimulate RNA primer synthesis by DNA primase <i>in vitro</i>. However, primase inhibits the Mcm4/6/7 helicase activity and this inhibition is abolished by the addition of competitor DNA. In contrast, the ATP hydrolysis activity of Mcm4/6/7 complex is not affected by primase. Mcm and primase proteins mutually stimulate their DNA-binding activities. Our findings indicate that a direct physical interaction between primase and Mcm proteins may facilitate priming reaction by the former protein, suggesting that efficient DNA synthesis through helicase-primase interactions may be conserved in eukaryotic chromosomes.</p></div

Effect of primase on DNA-binding activity of Mcm4/6/7 and Mcm2∼7.

<p>DNA-binding activities on ssDNA were examined in gel shift assays. (A) The proteins added were; 37.5 ng (lane 2), 75 ng (lane 3) and 150 ng (lane 4) of Mcm4/6/7; 75 ng (lane 5), 150 ng (lane 6), and 300 ng (lane 7) of Mcm2∼7. A constant amount of Mcm4/6/7 (15 ng/2.3 nM) in (B) or Mcm2∼7 (360 ng/50 nM) in (D) and various amounts of the p48/p58 primase and GINS were added. The proteins added were: 30, 60, and 120 ng of primase (25 nM, 50 nM and 100 nM, respectively, as a monomer) and 30, 60, 120, 250, and 500 ng of GINS (25 nM, 50 nM, 100 nM, 200 nM and 400 nM, respectively) in B, and 60 and 120 ng of primase in D. The reaction mixtures were incubated with 20 fmole labeled substrate (132 mer) at 30°C for 30 min, and were applied on a 5% native polyacrylamide gel (32.3:1) containing 1x TBE, followed by detection with autoradiography. In (C), a constant amount of Mcm4/6/7 (50 ng) or various amounts of Mcm4/6/7 (25 ng, 50 ng and 100 ng), and a constant amount of p48/p58 (50 ng) or various amounts of the p48/p58 primase (25 ng, 50 ng and 75 ng) were added. The DNA-binding reactions were separated, and two-thirds of reaction mixtures were incubated with oligonucleotide DNA (37mer-dT₅₀), and then the reactions were separated. A half was run in 5% native gel (acrylamide: bis = 37.5:1) containing 5% glycerol, 0.5x TBE (left), and the rest was run in 5% native gel (acrylamide: bis = 32.3:1) containing 1x TBE (right).</p

Direct interaction between Mcm assemblies/subunits and primase.

<p>(A) The list of the tag present in the Mcm2∼7 complex and p48/p58 or p48 protein used in (B), (C), (D), (E), and (F). The purified Mcm2∼7 complex (1 µg) was mixed with the His-tagged p48/p58 complex (0.25 µg) (B, D, and E), primase (0.25 µg) plus GINS proteins (1 µg) (C and D), or His-p48 (0.5 µg) (F), and immuno-precipitation was performed using anti-Flag M2 antibody beads (Sigma; Flag tag on Mcm5). The bound proteins were eluted with 0.1 M glycine (pH 2.8) (B, C, E, and F) or boiled with SDS sample buffer (D). The eluted samples (shown as “B”) and 1/10 (for silver-staining) or 1/20 (for Western blotting) of unbound fractions (shown as “U”) were analyzed as indicated. The heavy chain is visible in silver staining due to dissociation of the antibody from the beads in first elution (C). DNase I treatment was conducted in the samples without oligonucleotide to remove DNA that might be present in the purified protein fractions. (G) Extracts of Sf9 insect cells expressing the subunits of human DNA primase (p48 and p58) and the indicated Mcm protein were subjected to immuno-precipitation analyses using anti-Flag agarose beads (GE Healthcare). The Flag tag was on p58 in the two upper panels and on Mcm7 in the lower panel. Elution of bound proteins from the beads was carried out using a buffer containing the Flag peptide. For each sample, aliquots of the unbound and bound materials were analyzed by immuno-blotting using the indicated antibodies. Samples were run on 7.5% (B) or 4–20% gradient gel (C, D, E, and F). Star marker * in D indicated a non-special band.</p

Stimulation of RNA primer synthesis by Mcm complexes.

<p>RNA primer was synthesized by p48/p58 primase in the absence or presence of Mcm and/or GINS complex on poly(dT) (A-C) or on M13 mp18 ssDNA (D). RNA primer synthesis by DNA polymerase α-primase on M13 mp18 ssDNA substrate was examined in the absence or presence of Mcm complex (E). After incubation for 1 hr at 37°C, the products were applied on 20% denaturing polyacrylamide gel in 1x TBE buffer. A labeled oligonucleotide (dT_12–18) and 10-bp DNA ladder were used as a size marker. (A) The proteins added were; 100 ng (lanes 3–7, 9 and 10) and 300 ng (lane 2) of p48/58 primase; 100 ng (lane 4), 200 ng (lane 5), 400 ng (lane 6), and 600 ng (lanes 7 and 8) of Mcm4/6/7; or 200 ng (lane 9) and 400 ng (lanes 10 and 11) of Mcm2∼7. (B) The proteins added were; 100 ng (lanes 3–6, 8 and 9) and 300 ng (lane 2) of p48/58 primase; 100 ng (lane 4), 200 ng (lane 5) and 400 ng (lanes 6–9) of Mcm2∼7; and 0.5 µg (lane 8) and 1 µg (lane 9) of GINS. (C) The proteins added were; 100 ng of p48/58 (lanes 2–6) and 0.25 µg (lane 3), 0.5 µg (lane 4), 1 µg (lane 5) and 1.5 µg (lanes 6 and 7) of GINS. (D) The proteins added were; (lanes 2–11) 100 ng of p48/58 primase; 100 ng (lane 3), 200 ng (lane 4), 400 ng (lane 5), and 600 ng (lane 6) of Mcm4/6/7; or 50 ng (lane 11), 100 ng (lane 10), 150 ng (lane 9), and 200 ng (lanes 8) of Mcm2∼7. (E) The proteins added were; (lanes 2–8) 100 ng of human DNA polymerase α-primase complex; 100 ng (lane 3), 200 ng (lane 4), and 300 ng (lane 5) of Mcm4/6/7; or 50 ng (lane 6), 100 ng (lane 7), and 150 ng (lane 8) of Mcm2∼7. More than three independent experiments were carried out.</p

CDC28 phosphorylates Cac1p and regulates the association of chromatin assembly factor i with chromatin

<div><p>Chromatin Assembly Factor I (CAF-I) plays a key role in the replication-coupled assembly of nucleosomes. It is expected that its function is linked to the regulation of the cell cycle, but little detail is available. Current models suggest that CAF-I is recruited to replication forks and to chromatin via an interaction between its Cac1p subunit and the replication sliding clamp, PCNA, and that this interaction is stimulated by the kinase <i>CDC7</i>. Here we show that another kinase, <i>CDC28</i>, phosphorylates Cac1p on serines 94 and 515 in early S phase and regulates its association with chromatin, but not its association with PCNA. Mutations in the Cac1p-phosphorylation sites of <i>CDC28</i> but not of <i>CDC7</i> substantially reduce the <i>in vivo</i> phosphorylation of Cac1p. However, mutations in the putative <i>CDC7</i> target sites on Cac1p reduce its stability. The association of CAF-I with chromatin is impaired in a <i>cdc28–1</i> mutant and to a lesser extent in a <i>cdc7–1</i> mutant. In addition, mutations in the Cac1p-phosphorylation sites by both <i>CDC28</i> and <i>CDC7</i> reduce gene silencing at the telomeres. We propose that this phosphorylation represents a regulatory step in the recruitment of CAF-I to chromatin in early S phase that is distinct from the association of CAF-I with PCNA. Hence, we implicate <i>CDC28</i> in the regulation of chromatin reassembly during DNA replication. These findings provide novel mechanistic insights on the links between cell-cycle regulation, DNA replication and chromatin reassembly.</p></div