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

Lin, Yo-Chuen; Liu, Dazhen; Chakraborty, Arindam; Kadyrova, Lyudmila Y.; Shang, You; Hao, Qinyu; Mitra, Jaba; Hsu, Rosaline Y.C.; Arif, Mariam K.; Adusumilli, Sneha; Liao, Ting‐Wei; Ha, Taekjip; Kadyrov, Farid A.; Prasanth, Kannanganattu V.; Prasanth, Supriya G.

doi:10.1073/pnas.2121406119

Cited by 12 publications

(11 citation statements)

References 66 publications

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“…Although the deletion of either Orc1 or Orc2 has a profound effect on cell cycle progression (Okano-Uchida et al , 2018; Chou et al , 2021), Orc6 siRNA had a minimal effect on cell cycle progression in hTERT-RPE1 (Fig. 6, this study) and U2OS cells (Lin et al , 2022). These results suggest that incomplete ORC hexamers could recruit pre-RC to chromatin after the S phase in differentiated cells, although all subunits of the Orc1–6 hexamer are presumably essential in undifferentiated cells such as those in early embryogenesis (Prasanth et al , 2002; Ortega et al , 2016).…”

Section: Discussionmentioning

confidence: 54%

“…Orc1–5 exhibit structural similarities, whereas Orc6 shows no structural similarity to other ORC subunits (Duncker et al , 2009), implying functional dissimilarity. Orc6 has the potential to independently bind to DNA regardless of Orc1-5 (Xu et al , 2020), participate in DNA repair (Lin et al , 2022), and contribute to mitosis (Bernal & Venkitaraman, 2011; Semple et al , 2006; Balasov et al , 2009) in various living organisms. However, the function of human Orc6 during the cell cycle remains largely unknown.…”

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confidence: 99%

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Orc6 dissociation from chromatin prevents premature loading of MCM at G2 and tetraploid production

Hayashi-Takanaka,

Hiratani,

Haraguchi

et al. 2024

Preprint

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DNA replication is tightly regulated to occur only once per cell cycle. The untimely initiation of DNA replication can result in genome instability, leading to aneuploidy, which has been associated with early senescence and cancer. The pre-replication complex, comprising the origin recognition complex (ORC; Orc1-6), Cdc6, Cdt1, and MCM, is required for initiating DNA replication, although the function of Orc6 is yet to be elucidated. Here, we show that Orc6 dissociates from chromatin upon entering the S-phase and that the Orc6 dissociation depends on proteasome activity. Treatment that inhibits proteasome activity, which declines with aging, increases the senescence marker p21 levels, and promotes cell cycle arrest in human immortalized hTERT-RPE1 cells. This treatment induced large nuclei with high levels of chromatin-bound Orc6 and MCM without undergoing mitosis. When the proteasome activity recovered, those cells with high levels of chromatin-bound Orc6 and MCM proceeded to whole-genome DNA replication, confirming that they were tetraploid G1 cells. We propose that proteasome-dependent dissociation of Orc6 from chromatin after S-phase is essential for preventing MCM reloading and the subsequent development of tetraploid cells.

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Section: Discussionmentioning

confidence: 54%

mentioning

confidence: 99%

Orc6 dissociation from chromatin prevents premature loading of MCM at G2 and tetraploid production

Hayashi-Takanaka,

Hiratani,

Haraguchi

et al. 2024

Preprint

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“…5). It is unclear whether both pathways are used in vivo: on one hand a Meier-Gorlin Syndrome mutant mapping to the C-terminal alpha-helix of ORC6 that interacts with ORC3 and inhibits ORC6 interaction with ORC1-5 also inhibits MCM loading in Drosophila melanogaster 34 ; on the other hand, depletion of ORC6 from human U2OS cells was shown to have no effect on MCM loading levels in a single cell cycle 35 . ORC6-dependent loading may occur through formation of the ORC6-dependent hMO* complex.…”

Section: Discussionmentioning

confidence: 99%

MCM Double Hexamer Loading Visualised with Human Proteins

Weissmann,

Greiwe,

Pühringer

et al. 2024

Preprint

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Eukaryotic DNA replication begins with the loading of the MCM replicative DNA helicase as a head-to-head double hexamer (DH) at origins of DNA replication1–3. Our current understanding of how DH is assembled by the Origin Recognition Complex (ORC), CDC6 and CDT1 comes mostly from budding yeast. Here we characterise human DH (hDH) loading using biochemical reconstitution and cryo-electron microscopy with purified proteins. We show that hDH engages DNA differently from yeast (yDH), and generates ∼5 base pairs of unwound DNA at the interface between hexamers, as seen in hDH isolated from cells4. We identify several differences from yeast in the order of factor recruitment and dependencies during hDH assembly. Unlike yeast5–8, the ORC6 subunit of ORC is not essential for initial MCM recruitment or hDH loading, but contributes to an alternative hDH assembly pathway requiring an intrinsically disordered region (IDR) in ORC1, which may work through a novel MCM-ORC (hMO*) intermediate. Our work presents a detailed view of how DHs are assembled in an organism utilising sequence-independent replication origins, it provides further evidence for diversity in eukaryotic DH assembly mechanisms9, and it represents the first step toward reconstitution of DNA replication initiation with purified human proteins.

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“…ORC is now implicated in DNA repair and DDR. [ 50 ] It has previously been shown that ORC mutations result in arrested forks below the threshold required for activating the S‐phase checkpoint. [ 122 ] Similarly, ORC dysregulation resulting in apoptosis, possibly due to overactive checkpoint activation, has been documented.…”

Section: The Role Of Other Pre‐rc Components In Maintaining Genome St...mentioning

confidence: 99%

“…[ 47–49 ] Recently, our results prompted the possibility that hOrc6 is dispensable for MCM loading and is not needed for pre‐RC establishment in human cells. [ 50 ] Instead, it plays a crucial role in replication progression in S‐phase and facilitates mismatch repair (MMR) during DNA replication. The “enigmatic nature” of human Orc6 raises the question, “Is hOrc6 a component of ORC?” “What is the biochemical role of the sub‐stoichiometric amounts of hOrc6 in the Orc1‐5 complex?”…”

Section: Introductionmentioning

confidence: 99%

ORChestra coordinates the replication and repair music

2023

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

Cited by 12 publications

References 66 publications

Orc6 dissociation from chromatin prevents premature loading of MCM at G2 and tetraploid production

Orc6 dissociation from chromatin prevents premature loading of MCM at G2 and tetraploid production

MCM Double Hexamer Loading Visualised with Human Proteins

ORChestra coordinates the replication and repair music

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