Reconstitution of DNMT1 complex with hemimethylated DNA, doubly monoubiquitinated PAF15, and PCNA for structural analysis
Por:
A. RUIZ-ALBOR, B. CHAVES-ARQUERO, M. FERRERAS-GUTIÉRREZ, M. BARBERA, A. GONZÁLEZ-MAGAÑA, R. NÚÑEZ-RAMÍREZ, A. DE BIASIO and F. BLANCO
Publicada:
1 ago 2025
Ahead of Print:
1 jun 2025
Resumen:
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.
Filiaciones:
A. RUIZ-ALBOR:
CSIC, Inst Biomed Valencia IBV, Valencia 46010, Spain
B. CHAVES-ARQUERO:
CSIC, Ctr Invest Biol Margarita Salas CIB, Madrid 28040, Spain
M. FERRERAS-GUTIÉRREZ:
CSIC, Ctr Invest Biol Margarita Salas CIB, Madrid 28040, Spain
M. BARBERA:
CSIC, Ctr Invest Biol Margarita Salas CIB, Madrid 28040, Spain
A. GONZÁLEZ-MAGAÑA:
Univ Basque Country, CSIC, Inst Biofis, Leioa 48940, Spain
R. NÚÑEZ-RAMÍREZ:
CSIC, Ctr Invest Biol Margarita Salas CIB, Madrid 28040, Spain
A. DE BIASIO:
King Abdullah Univ Sci & Technol, Thuwal 23955, Saudi Arabia
:
CSIC, Inst Biomed Valencia IBV, Valencia 46010, Spain
Ctr Invest Principe Felipe CIPF, Associated Unit IBV, Valencia 46012, Spain
Green Accepted, hybrid
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