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1. The human origin recognition complex is essential for pre-RC assembly, mitosis, and maintenance of nuclear structure

   https://doi.org/10.7554/eLife.61797  

   Chou, Hsiang-Chen; Bhalla, Kuhulika; Demerdesh, Osama EL; Klingbeil, Olaf; Hanington, Kaarina; Aganezov, Sergey; Andrews, Peter; Alsudani, Habeeb; Chang, Kenneth; Vakoc, Christopher R; et al (February 2021, eLife)

   null (Ed.)

   The origin recognition complex (ORC) cooperates with CDC6, MCM2-7, and CDT1 to form pre-RC complexes at origins of DNA replication. Here, using tiling-sgRNA CRISPR screens, we report that each subunit of ORC and CDC6 is essential in human cells. Using an auxin-inducible degradation system, we created stable cell lines capable of ablating ORC2 rapidly, revealing multiple cell division cycle phenotypes. The primary defects in the absence of ORC2 were cells encountering difficulty in initiating DNA replication or progressing through the cell division cycle due to reduced MCM2-7 loading onto chromatin in G1 phase. The nuclei of ORC2-deficient cells were also large, with decompacted heterochromatin. Some ORC2-deficient cells that completed DNA replication entered into, but never exited mitosis. ORC1 knockout cells also demonstrated extremely slow cell proliferation and abnormal cell and nuclear morphology. Thus, ORC proteins and CDC6 are indispensable for normal cellular proliferation and contribute to nuclear organization. 

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2. Conservation of a DNA Replication Motif among Phylogenetically Distant Budding Yeast Species

   https://doi.org/10.1093/gbe/evab137  

   Maria, Haniam; Kapoor, Shivali; Liu, Tao; Rusche, Laura N (July 2021, Genome Biology and Evolution)

   Wolfe, Kenneth (Ed.)

   Abstract Eukaryotic DNA replication begins at genomic loci termed origins, which are bound by the origin recognition complex (ORC). Although ORC is conserved across species, the sequence composition of origins is more varied. In the budding yeast Saccharomyces cerevisiae, the ORC-binding motif consists of an A/T-rich 17 bp “extended ACS” sequence adjacent to a B1 element composed of two 3-bp motifs. Similar sequences occur at origins in closely related species, but it is not clear when this type of replication origin arose and whether it predated a whole-genome duplication that occurred around 100 Ma in the budding yeast lineage. To address these questions, we identified the ORC-binding sequences in the nonduplicated species Torulaspora delbrueckii. We used chromatin immunoprecipitation followed by sequencing and identified 190 ORC-binding sites distributed across the eight T. delbrueckii chromosomes. Using these sites, we identified an ORC-binding motif that is nearly identical to the known motif in S. cerevisiae. We also found that the T. delbrueckii ORC-binding sites function as origins in T. delbrueckii when cloned onto a plasmid and that the motif is required for plasmid replication. Finally, we compared an S. cerevisiae origin with two T. delbrueckii ORC-binding sites and found that they conferred similar stabilities to a plasmid. These results reveal that the ORC-binding motif arose prior to the whole-genome duplication and has been maintained for over 100 Myr. 

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3. Orc6 is a component of the replication fork and enables efficient mismatch repair

   https://doi.org/10.1073/pnas.2121406119  

   Lin, Yo-Chuen; Liu, Dazhen; Chakraborty, Arindam; Kadyrova, Lyudmila Y.; Song, You Jin; Hao, Qinyu; Mitra, Jaba; Hsu, Rosaline Y.; Arif, Mariam K.; Adusumilli, Sneha; et al (May 2022, Proceedings of the National Academy of Sciences)

   In eukaryotes, the origin recognition complex (ORC) is required for the initiation of DNA replication. The smallest subunit of ORC, Orc6, is essential for prereplication complex (pre-RC) assembly and cell viability in yeast and for cytokinesis in metazoans. However, unlike other ORC components, the role of human Orc6 in replication remains to be resolved. Here, we identify an unexpected role for hOrc6, which is to promote S-phase progression after pre-RC assembly and DNA damage response. Orc6 localizes at the replication fork and is an accessory factor of the mismatch repair (MMR) complex. In response to oxidative damage during S phase, often repaired by MMR, Orc6 facilitates MMR complex assembly and activity, without which the checkpoint signaling is abrogated. Mechanistically, Orc6 directly binds to MutSα and enhances the chromatin-association of MutLα, thus enabling efficient MMR. Based on this, we conclude that hOrc6 plays a fundamental role in genome surveillance during S phase. 

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4. Cell size regulation in bacteria: a tale of old regulators with new mechanisms

   https://doi.org/10.1101/2025.02.22.639668  

   Khan, Ezza; Mera, Paola E (February 2025, bioRxiv)

   SUMMARY Proper function in a bacterial cell relies on intrinsic cell size regulation. The molecular mechanisms underlying how bacteria maintain their cell size remain unclear. The conserved regulator DnaA, the initiator of chromosome replication, is associated to size regulation by controlling the number of origins of replication (oriC) per cell. In this study, we identify and characterize a new mechanism in which DnaA modulates cell size independently oforiC-copy number. By altering the levels of DnaA without impacting chromosome replication, we demonstrate that DnaA’s activity as a transcription factor can slow down cell elongation rate resulting in cells that are ∼20% smaller. We identify the peptidoglycan biosynthetic enzyme MurD as a key player of cell size regulation inCaulobacter crescentusand in the evolutionarily distant bacteriumEscherichia coli. Collectively, our findings provide mechanistic insights to the complex regulation of cell size in bacteria. 

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5. Chromatin’s Influence on Pre-Replication Complex Assembly and Function

   https://doi.org/10.3390/biology13030152  

   Ahmad, Hina; Chetlangia, Neha; Prasanth, Supriya G (March 2024, Biology)

   In all eukaryotes, the initiation of DNA replication requires a stepwise assembly of factors onto the origins of DNA replication. This is pioneered by the Origin Recognition Complex, which recruits Cdc6. Together, they bring Cdt1, which shepherds MCM2-7 to form the OCCM complex. Sequentially, a second Cdt1-bound hexamer of MCM2-7 is recruited by ORC-Cdc6 to form an MCM double hexamer, which forms a part of the pre-RC. Although the mechanism of ORC binding to DNA varies across eukaryotes, how ORC is recruited to replication origins in human cells remains an area of intense investigation. This review discusses how the chromatin environment influences pre-RC assembly, function, and, eventually, origin activity. 

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