Biochemical and Biophysical Research Communications
Structure of PCNA in complex with DNMT1 PIP box reveals the basis for the molecular mechanism of the interaction
Introduction
Cytosine methylation in CpG dinucleotides is one of the major epigenetic marks in vertebrates [1,2]. DNA methylation is involved in retrotransposon silencing, genome imprinting, X chromosome inactivation and carcinogenesis, and contributes to the establishment of tissue-specific gene expression patterns [3]. Importantly, in addition to genomic information, DNA methylation patterns are faithfully inherited in each cycle of DNA replication to maintain cell identity [4]. Three DNA methyltransferases have been identified in mammals, DNMT1, DNMT3A and DNMT3B [5,6]. Among of them, DNMT1 preferentially methylates hemimethylated DNA where only the parent strand CpG is methylated, thus functioning as a maintenance DNA methyltransferase [7]. Ubiquitin-like, containing PHD and RING finger domains, 1 (UHRF1, also known as ICBP90 and NP95) plays a pivotal role in DNA methylation maintenance [8,9]: the SET and RING associated (SRA) domain of UHRF1 specifically recognizes hemimethylated DNA [[10], [11], [12]], and consequently the really interesting new gene (RING) domain of UHRF1 catalyzes the multi mono-ubiquitination of histone H3 at K14, K18 and K23 [[13], [14], [15]]. The multi-mono-ubiquitylated histone H3 recruits DNMT1 to the methylation sites and enhances the DNA methylation activity of DNMT1 [15], and finally unmethylated CpG sequence in nascent strand is remethylated.
Proliferating cell nuclear antigen (PCNA) is an essential factor for DNA replication, repair and cell cycle regulation, and functions as a DNA clamp and a hub protein that physically interacts with approximately one hundred proteins including polymerases, ligases, DNA modifying enzymes and epigenetic factors [16]. Recently, replication factors have emerged as regulators of DNA methylation maintenance. DNA ligase 1 (LIG1) harboring methylated K126 directly interacts with UHRF1 and recruits it to replication sites to regulate DNA methylation maintenance [17,18]. DNMT1 and UHRF1 accumulate at replication sites that are characterized by PCNA dense region [8,9]. In particular, DNMT1 has a PCNA binding motif (PIP box; PCNA interacting protein) at the flexible N-terminal region (164QTTITSHF171) [19], and accumulates at replication sites with PCNA during early and mid S-phase [20]. Interestingly, the consensus sequence of the PIP box motif is Qxxhxxaa, where “x”, “h” and “a” indicate any residue, a hydrophobic residue and an aromatic residue, respectively, whereas the C-terminal aromatic residues of the DNMT1 PIP box motif are replaced with His-Phe residues, which have not been observed in the PIP box motifs of other PCNA interacting partners [16]. However, the molecular mechanism by which DNMT1 interacts with PCNA through the atypical PIP box motif remains unknown.
In the present study, we analyzed the interaction between PCNA and the DNMT1 PIP box peptide by a biochemical assay and determined the crystal structure of PCNA in complex with the DNMT1 PIP box peptide. Our crystallographic analysis revealed the detailed interaction between DNMT1 and a PCNA and novel intra-interaction within the PIP box peptide.
Section snippets
Peptide preparation
DNMT1 PIP box peptide, NH2-161STRQTTITSHFAKGPAKRKP180-COOH, and the candidate PIP box motif of UHRF1, NH2-776QPLQTVLNQLFPGYGNGR793-COOH, were synthesized by Toray Research Center (Tokyo, Japan).
Protein expression and purification
The gene encoding human PCNA was amplified by PCR with Human Universal QUICK-Clone™ (Clontech). The amplified DNA was cloned into the pET47 vector (Novagen) by the In-Fusion™ cloning method (Clontech). Six-histidine-tagged PCNA was expressed in E. coli Rosetta 2 (DE3) (Novagen) cells. Cells were grown at
Biochemical assay for detecting the interaction between PCNA and DNMT1
Given that DNMT1 binds to PCNA via its PIP box motif in mammalian cells, we tested whether DNMT1 PIP box peptide (hereafter PIPDNMT1), residues 161–180, could bind to PCNA in vitro. Recombinant human PCNA was prepared by expression in E. coli and highly purified. Isothermal titration calorimetry (ITC) data showed that PIPDNMT1 bound to PCNA with Kd of 1.00 ± 0.05 μM (Fig. 1A), which is a binding affinity comparable to those between PCNA and the PIP box motifs of other interacting proteins (
Discussion
DNMT1 and UHRF1, which are essential for DNA methylation maintenance, have been shown to localize at the replication sites during early and mid S-phase, in which PCNA functions as a binding platform for replication and repair factors. Our results provide novel insight into the interaction between PCNA and the DNMT1 PIP box. The conformation of the DNMT1 PIP box when bound to PCNA is nearly identical to that of other PIP box motifs; however, the DNMT1 PIP box forms an intramolecular interaction
Author contributions
K.A. conceived the study and experimental design, analyzed experiments and wrote the manuscript. T.J. and R.M. purified recombinant protein. S.K. performed ITC experiments and co-wrote the manuscript. R.M. performed the crystallization of the complex and K.A. analyzed the structure.
Accession number
The crystal structures of the PCNA in complex with DNMT1 PIP box peptide have been deposited in the Protein Data Bank under accession code 6K3A.
Conflict of interest statement
None declared.
Acknowledgements
We would like to thank the beamline staff at the Photon Factory for X-ray data collection. We also thank Dr. A. Hishiki for discussion of this paper. This study was supported by a PRESTO (14530337) from JST and MEXT, Grant-in-Aid for Scientific Research (B), 18H02392, (K.A.).
References (31)
- et al.
Methylation-induced repression--belts, braces, and chromatin
Cell
(1999) - et al.
Processive methylation of hemimethylated CpG sites by mouse Dnmt1 DNA methyltransferase
J. Biol. Chem.
(2005) - et al.
PCNA, the maestro of the replication fork
Cell
(2007) - et al.
Structure insights into the molecular mechanism of the interaction between UHRF2 and PCNA
Biochem. Biophys. Res. Commun.
(2017) - et al.
Structural basis for novel interactions between human translesion synthesis polymerases and proliferating cell nuclear antigen
J. Biol. Chem.
(2009) - et al.
Crystal structure of human PCNA in complex with the PIP box of DVC1
Biochem. Biophys. Res. Commun.
(2016) - et al.
Structural and Thermodynamic Analysis of Human PCNA with Peptides Derived from DNA Polymerase-δ p66 Subunit and Flap Endonuclease-1
Structure
(2004) DNA methylation patterns and epigenetic memory
Genes Dev.
(2002)- et al.
DNA methylation in mammals
Cold Spring Harbor Perspect. Biol.
(2014) - et al.
Programming of DNA methylation patterns
Annu. Rev. Biochem.
(2012)
Cloning and characterization of a family of novel mammalian DNA (cytosine-5) methyltransferases
Nat. Genet.
Enzymatic properties of de novo-type mouse DNA (cytosine-5) methyltransferases
Nucleic Acids Res.
The SRA protein Np95 mediates epigenetic inheritance by recruiting Dnmt1 to methylated DNA
Nature
UHRF1 plays a role in maintaining DNA methylation in mammalian cells
Science (New York, N.Y.).
Structural basis for recognition of hemi-methylated DNA by the SRA domain of human UHRF1
Nature
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