Reconstitution of DNMT1 complex with hemimethylated DNA, doubly monoubiquitinated PAF15, and PCNA for structural analysis
Eukaryotic cells use multiprotein complexes to make a copy of their chromosomes. Many of those complexes assemble on Proliferating Cell Nuclear Antigen (PCNA), a toroidal homotrimer that embraces the DNA duplex. The enzyme DNA Methyltransferase 1 (DNMT1) methylates cytosine bases in the daughter DNA...
| Authors: | , , , , , , , |
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| Format: | article |
| Status: | Published version |
| Publication Date: | 2025 |
| Country: | España |
| Institution: | Centro de Investigación Principe Felipe (CIPF) |
| Repository: | r-CIPF. Repositorio Institucional Producción Científica del Centro de Investigación Principe Felipe (CIPF) |
| OAI Identifier: | oai:cipf.fundanetsuite.com:p4567 |
| Online Access: | https://cipf.fundanetsuite.com/Publicaciones/ProdCientif/PublicacionFrw.aspx?id=4567 |
| Access Level: | Open access |
| Keyword: | DNMT1 PCNA PAF15 |
| Summary: | Eukaryotic cells use multiprotein complexes to make a copy of their chromosomes. Many of those complexes assemble on Proliferating Cell Nuclear Antigen (PCNA), a toroidal homotrimer that embraces the DNA duplex. The enzyme DNA Methyltransferase 1 (DNMT1) methylates cytosine bases in the daughter DNA strand, replicating the methylation pattern of the parental one. DNMT1 is recruited to the replication fork by the regulatory protein p15 when it is doubly monoubiquitinated in its N-terminal disordered tail, and the central region of p15 binds to the front face of the PCNA ring. We have reconstituted the complex formed by DNMT1, a DNA duplex with a methylated cytosine, doubly monoubiquitinated p15, and PCNA from the isolated components. A complex with equimolar stoichiometry is detected in solution by mass photometry, and particles with the expected size are observed in negatively stained electron micrographs but dissociate upon vitrification. The structure of the complex predicted with AlphaFold is consistent with the available experimental information, providing a plausible model for DNMT1 action anchored to PCNA, and suggests that methylation of the newly synthesized DNA strand can occur in concert with lagging strand replication by DNA polymerase delta. |
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