Structure of PCNA in complex with DNMT1 PIP box reveals the basis for the molecular mechanism of the interaction

https://doi.org/10.1016/j.bbrc.2019.06.060Get rights and content

Highlights

  • The crystal structure of PCNA in complex with the DNMT1 PIP box motif is solved.

  • Conserved residues in the DNMT1 PIP box are recognized by the Q- and hydrophobic pockets of PCNA.

  • Intramolecular interactions within the PIP box motif provide a possible stabilization mechanism for the motif conformation.

Abstract

DNMT1 is a C5-DNA methyltransferase that plays a pivotal role in DNA methylation maintenance. During early and mid S-phase, DNMT1 accumulates at DNA replication sites by binding to proliferating cell nuclear antigen (PCNA), an essential factor for DNA replication, through a PIP box motif. However, the molecular mechanism by which the DNMT1 PIP box motif binds to PCNA remains unclear. Here, we report the crystal structure of PCNA bound to DNMT1 PIP box peptide. The structure reveals the detailed interaction between PCNA and DNMT1 PIP box; conserved glutamine and hydrophobic/aromatic residues in the PIP box are recognized by the Q- and hydrophobic pockets of PCNA, respectively. The structure also shows novel intramolecular interactions within the PIP box motif, which stabilize the helix conformation in the PIP box. Our data provide structural insight into the recruitment of DNMT1 to replication sites by PCNA.

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.).

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