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1. 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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2. 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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3. ORChestra coordinates the replication and repair music

   https://doi.org/10.1002/bies.202200229  

   Liu, Dazhen; Sonalkar, Jay; Prasanth, Supriya G. (February 2023, BioEssays)

   Abstract Error‐free genome duplication and accurate cell division are critical for cell survival. In all three domains of life, bacteria, archaea, and eukaryotes, initiator proteins bind replication origins in an ATP‐dependent manner, play critical roles in replisome assembly, and coordinate cell‐cycle regulation. We discuss how the eukaryotic initiator, Origin recognition complex (ORC), coordinates different events during the cell cycle. We propose that ORC is the maestro driving the orchestra to coordinately perform the musical pieces of replication, chromatin organization, and repair. 

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4. Single-molecule imaging reveals the mechanism of bidirectional replication initiation in metazoa

   https://doi.org/10.1016/j.cell.2024.05.024  

   Terui, Riki; Berger, Scott E; Sambel, Larissa A; Song, Dan; Chistol, Gheorghe (July 2024, Cell)

   Behie, Scott (Ed.)

   Metazoan genomes are copied bidirectionally from thousands of replication origins. Replication initiation entails the assembly and activation of two CMG helicases (Cdc45⋅Mcm2-7⋅GINS) at each origin. This requires several replication firing factors (including TopBP1, RecQL4, and DONSON) whose exact roles are still under debate. How two helicases are correctly assembled and activated at each origin is a long-standing question. By visualizing the recruitment of GINS, Cdc45, TopBP1, RecQL4, and DONSON in real time, we uncovered that replication initiation is surprisingly dynamic. First, TopBP1 transiently binds to the origin and dissociates before the start of DNA synthesis. Second, two Cdc45 are recruited together, even though Cdc45 alone cannot dimerize. Next, two copies of DONSON and two GINS simultaneously arrive at the origin, completing the assembly of two CMG helicases. Finally, RecQL4 is recruited to the CMG⋅DONSON⋅DONSON⋅CMG complex and promotes DONSON dissociation and CMG activation via its ATPase activity. 

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5. 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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