A dual role for the chromatin reader ORCA/LRWD1 in targeting the origin recognition complex to chromatin

• Published in: The EMBO journal 2023-09, Vol.42 (18), p.e114654
• Main Authors: Sahu, Sumon, Ekundayo, Babatunde E, Kumar, Ashish, Bleichert, Franziska
• Format: Article
• Language: English
• Subjects: Animals; Assembly; Cages; Chromatin; Chromatin - genetics; Compaction; Conserved sequence; Cryoelectron Microscopy; Deoxyribonucleic acid; DNA; DNA biosynthesis; DNA Replication; DNA structure; Electron microscopy; EMBO09; EMBO13; Heterochromatin; Heterochromatin - genetics; Histones; Initiators; Life Sciences; Mammalian cells; Mammals - genetics; Methylation; Nucleosomes; Nucleosomes - genetics; Nucleotide sequence; Origin recognition complex; Origin Recognition Complex - genetics; Origin Recognition Complex - metabolism; Proteins; Recognition; Replication; Replication initiation; Replication Origin; Replication origins; Transcription Factors - genetics
• ISSN: 0261-4189; 1460-2075; 1460-2075
• DOI: 10.15252/embj.2023114654

Eukaryotic cells use chromatin marks to regulate the initiation of DNA replication. The origin recognition complex (ORC)‐associated protein ORCA plays a critical role in heterochromatin replication in mammalian cells by recruiting the initiator ORC, but the underlying mechanisms remain unclear. Here, we report crystal and cryo‐electron microscopy structures of ORCA in complex with ORC's Orc2 subunit and nucleosomes, establishing that ORCA orchestrates ternary complex assembly by simultaneously recognizing a highly conserved peptide sequence in Orc2, nucleosomal DNA, and repressive histone trimethylation marks through an aromatic cage. Unexpectedly, binding of ORCA to nucleosomes prevents chromatin array compaction in a manner that relies on H4K20 trimethylation, a histone modification critical for heterochromatin replication. We further show that ORCA is necessary and sufficient to specifically recruit ORC into chromatin condensates marked by H4K20 trimethylation, providing a paradigm for studying replication initiation in specific chromatin contexts. Collectively, our findings support a model in which ORCA not only serves as a platform for ORC recruitment to nucleosomes bearing specific histone marks but also helps establish a local chromatin environment conducive to subsequent MCM2‐7 loading. Synopsis Eukaryotic DNA replication relies on the initiator ORC to load replicative helicases onto DNA, but how this outcome is achieved in condensed chromatin remains unclear. Biochemical and structural studies uncover a dual mechanism for how the ORC‐associated protein ORCA supports ORC during replication initiation in chromatin. Crystal and cryo‐EM structures of ORCA in complex with ORC's Orc2 subunit and nucleosomes resolve molecular details of ternary complex assembly. ORCA uses an aromatic cage in its WD40 domain to recognize repressive histone trimethylation marks, while adjacent protein regions help stabilize ORCA on nucleosomes and chromatin. ORCA recruits ORC to chromatin marked by H4K20me3 in vitro . ORCA reduces self‐association of H4K20me3‐modified but not unmodified nucleosomes and diminishes H4K20me3‐chromatin compaction. These findings support a model in which ORCA not only recruits ORC to chromatin in a histone modification‐specific manner but also reorganizes local chromatin structure in preparation for DNA replication initiation. Graphical Abstract Biochemical and structural studies suggest that ORCA not only recruits ORC to chromatin with specifi