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

Non-Enzymatic DNA Cleavage Reaction Induced by 5-Ethynyluracil in Methylamine Aqueous Solution and Application to DNA Concatenation

<div><p>DNA can be concatenated by hybridization of DNA fragments with protruding single-stranded termini. DNA cleavage occurring at a nucleotide containing a DNA base analogue is a useful method to obtain DNA with designed protruding termini. Here, we report a novel non-enzymatic DNA cleavage reaction for DNA concatenation. We found that DNA is cleaved at a nucleotide containing 5-ethynyluracil in a methylamine aqueous solution to generate 5′-phosphorylated DNA fragment as a cleavage product. We demonstrated that the reaction can be applied to DNA concatenation of PCR-amplified DNA fragments. This novel non-enzymatic DNA cleavage reaction is a simple practical approach for DNA concatenation.</p></div

Chemical structures of thymine (T) and 5-ethynyluracil (EU).

<p>Chemical structures of thymine (T) and 5-ethynyluracil (EU).</p

Chemical formula of the DNA cleavage reaction.

<p>R is expected to be an abasic sugar derivative.</p

Construction of plasmid from two PCR-amplified DNA fragments.

<p>(A) Scheme of plasmid construction. (B) Primer sequences used for PCR. The two sequences underlined in red and blue are complementary to each other. (C–G) Pictures of agarose gel electrophoresis. (C) PCR-amplified DNA fragments 1.5 (lane 2) and 2.2 kbp (lane 3). (D) 1.5 and 2.2 kbp DNA fragments before (lane 2,3) and after DNA cleavage at 25°C for 48 h (lane 4,5), 37°C for 10 h (lane 6,7), and 70°C for 0.5 h (lane 8,9). MeNH₂ was removed from the samples by speed-vac before electrophoresis. (E) Hybridized 1.5 and 2.2 kbp DNA fragments derived from those without cleavage reaction (lane 2) and cleaved at 25°C for 48 h (lane 3), 37°C for 10 h (lane 4), and 70°C for 0.5 h (lane 5). (F,G) Intact purified plasmids (F) and EcoRV-digested plasmids (G) derived from the DNA fragments cleaved at 25°C for 48 h (lane 2,3), 37°C for 10 h (lane 4–6), and 70°C for 0.5 h (lane 7–9). (H) Sequencing results of primer-derived regions of the plasmids. Underlined letters correspond to EU in the primers.</p

Degradation of DNA oligonucleotides containing 5-ethynyluracil.

<p>(A), (B) HPLC charts of T₆(EU)T₆ before (gray) and after (black) the reaction in 14% NH₃aq (A) or 20% MeNH₂aq (B) at 70°C for 2 hours. (C), (D) (EU)T₂AT₂GT₂ (C) and T₂AT₂GT₂(EU)T (D) before (gray) and after (black) the reaction in 20% MeNH₂aq at 70°C for 2 hours.</p