Structural Mechanisms for Replicating DNA in Eukaryotes.

The faithful and timely copying of DNA by molecular machines known as replisomes depends on a disparate suite of enzymes and scaffolding factors working together in a highly orchestrated manner. Large, dynamic protein-nucleic acid assemblies that selectively morph between distinct conformations and compositional states underpin this critical cellular process. In this article, we discuss recent progress outlining the physical basis of replisome construction and progression in eukaryotes.

Yeast ORC sumoylation status fine-tunes origin licensing.

Sumoylation is emerging as a posttranslation modification important for regulating chromosome duplication and stability. The origin recognition complex (ORC) that directs DNA replication initiation by loading the MCM replicative helicase onto origins is sumoylated in both yeast and human cells. However, the biological consequences of ORC sumoylation are unclear. Here we report the effects of hypersumoylation and hyposumoylation of yeast ORC on ORC activity and origin function using multiple approaches. ORC hypersumoylation preferentially reduced the function of a subset of early origins, while Orc2 hyposumoylation had an opposing effect. Mechanistically, ORC hypersumoylation reduced MCM loading in vitro and diminished MCM chromatin association in vivo. Either hypersumoylation or hyposumoylation of ORC resulted in genome instability and the dependence of yeast on other genome maintenance factors, providing evidence that appropriate ORC sumoylation levels are important for cell fitness. Thus, yeast ORC sumoylation status must be properly controlled to achieve optimal origin function across the genome and genome stability.

A composite DNA element that functions as a maintainer required for epigenetic inheritance of heterochromatin.

Epigenetic inheritance of heterochromatin requires DNA-sequence-independent propagation mechanisms, coupling to RNAi, or input from DNA sequence, but how DNA contributes to inheritance is not understood. Here, we identify a DNA element (termed "maintainer") that is sufficient for epigenetic inheritance of pre-existing histone H3 lysine 9 methylation (H3K9me) and heterochromatin in Schizosaccharomyces pombe but cannot establish de novo gene silencing in wild-type cells. This maintainer is a composite DNA element with binding sites for the Atf1/Pcr1 and Deb1 transcription factors and the origin recognition complex (ORC), located within a 130-bp region, and can be converted to a silencer in cells with lower rates of H3K9me turnover, suggesting that it participates in recruiting the H3K9 methyltransferase Clr4/Suv39h. These results suggest that, in the absence of RNAi, histone H3K9me is only heritable when it can collaborate with maintainer-associated DNA-binding proteins that help recruit the enzyme responsible for its epigenetic deposition.

How bacteria initiate DNA replication comes into focus.

The ability to initiate DNA replication is a critical step in the proliferation of all organisms. In bacteria, this process is mediated by an ATP-dependent replication initiator protein, DnaA, which recognizes and melts replication origin (oriC) elements. Despite decades of biochemical and structural work, a mechanistic understanding of how DnaA recognizes and unwinds oriC has remained enigmatic. A recent study by Pelliciari et al. provides important new structural insights into how DnaA from Bacillus subtilis recognizes and processes its cognate oriC, showing how DnaA uses sequence features encoded in the origin to engage melted DNA. Comparison of the DnaA-oriC structure with archaeal/eukaryl replication origin complexes based on Orc-family proteins reveals a high degree of similarity in origin engagement by initiators from di domains of life, despite fundamental differences in origin melting mechanisms. These findings provide valuable insights into bacterial replication initiation and highlight the intriguing evolutionary history of this fundamental biological process.

Regulation of replication origin licensing by ORC phosphorylation reveals a two-step mechanism for Mcm2-7 ring closing.

Eukaryotic DNA replication must occur exactly once per cell cycle to maintain cell ploidy. This outcome is ensured by temporally separating replicative helicase loading (G1 phase) and activation (S phase). In budding yeast, helicase loading is prevented outside of G1 by cyclin-dependent kinase (CDK) phosphorylation of three helicase-loading proteins: Cdc6, the Mcm2-7 helicase, and the origin recognition complex (ORC). CDK inhibition of Cdc6 and Mcm2-7 is well understood. Here we use single-molecule assays for multiple events during origin licensing to determine how CDK phosphorylation of ORC suppresses helicase loading. We find that phosphorylated ORC recruits a first Mcm2-7 to origins but prevents second Mcm2-7 recruitment. The phosphorylation of the Orc6, but not of the Orc2 subunit, increases the fraction of first Mcm2-7 recruitment events that are unsuccessful due to the rapid and simultaneous release of the helicase and its associated Cdt1 helicase-loading protein. Real-time monitoring of first Mcm2-7 ring closing reveals that either Orc2 or Orc6 phosphorylation prevents Mcm2-7 from stably encircling origin DNA. Consequently, we assessed formation of the MO complex, an intermediate that requires the closed-ring form of Mcm2-7. We found that ORC phosphorylation fully inhibits MO complex formation and we provide evidence that this event is required for stable closing of the first Mcm2-7. Our studies show that multiple steps of helicase loading are impacted by ORC phosphorylation and reveal that closing of the first Mcm2-7 ring is a two-step process started by Cdt1 release and completed by MO complex formation.

Changing protein-DNA interactions promote ORC binding-site exchange during replication origin licensing.

During origin licensing, the eukaryotic replicative helicase Mcm2-7 forms head-to-head double hexamers to prime origins for bidirectional replication. Recent single-molecule and structural studies revealed that one molecule of the helicase loader ORC (origin recognition complex) can sequentially load two Mcm2-7 hexamers to ensure proper head-to-head helicase alignment. To perform this task, ORC must release from its initial high-affinity DNA-binding site and "flip" to bind a weaker, inverted DNA site. However, the mechanism of this binding-site switch remains unclear. In this study, we used single-molecule Förster resonance energy transfer to study the changing interactions between DNA and ORC or Mcm2-7. We found that the loss of DNA bending that occurs during DNA deposition into the Mcm2-7 central channel increases the rate of ORC dissociation from DNA. Further studies revealed temporally controlled DNA sliding of helicase-loading intermediates and that the first sliding complex includes ORC, Mcm2-7, and Cdt1. We demonstrate that sequential events of DNA unbending, Cdc6 release, and sliding lead to a stepwise decrease in ORC stability on DNA, facilitating ORC dissociation from its strong binding site during site switching. In addition, the controlled sliding we observed provides insight into how ORC accesses secondary DNA-binding sites at different locations relative to the initial binding site. Our study highlights the importance of dynamic protein-DNA interactions in the loading of two oppositely oriented Mcm2-7 helicases to ensure bidirectional DNA replication.

ORC1 enhances repressive epigenetic modifications on HIV-1 LTR to promote HIV-1 latency.

The human immunodeficiency virus type 1 (HIV-1) reservoir consists of latently infected cells which present a major obstacle to achieving a functional cure for HIV-1. The formation and maintenance of HIV-1 latency have been extensively studied, and latency-reversing agents (LRAs) that can reactivate latent HIV-1 by targeting the involved host factors are developed; however, their clinical efficacies remain unsatisfactory. Therefore, it is imperative to identify novel targets for more potential candidates or better combinations for LRAs. In this study, we utilized CRISPR affinity purification in situ of regulatory elements system to screen for host factors associated with the HIV-1 long terminal repeat region that could potentially be involved in HIV-1 latency. We successfully identified that origin recognition complex 1 (ORC1), the largest subunit of the origin recognition complex, contributes to HIV-1 latency in addition to its function in DNA replication initiation. Notably, ORC1 is enriched on the HIV-1 promoter and recruits a series of repressive epigenetic elements, including DNMT1 and HDAC1/2, and histone modifiers, such as H3K9me3 and H3K27me3, thereby facilitating the establishment and maintenance of HIV-1 latency. Moreover, the reactivation of latent HIV-1 through ORC1 depletion has been confirmed across various latency cell models and primary CD4+ T cells from people living with HIV-1. Additionally, we comprehensively validated the properties of liquid-liquid phase separation (LLPS) of ORC1 from multiple perspectives and identified the key regions that promote the formation of LLPS. This property is important for the recruitment of ORC1 to the HIV-1 promoter. Collectively, these findings highlight ORC1 as a potential novel target implicated in HIV-1 latency and position it as a promising candidate for the development of novel LRAs. IMPORTANCE: Identifying host factors involved in maintaining human immunodeficiency virus type 1 (HIV-1) latency and understanding their mechanisms prepares the groundwork to discover novel targets for HIV-1 latent infection and provides further options for the selection of latency-reversing agents in the "shock" strategy. In this study, we identified a novel role of the DNA replication factor origin recognition complex 1 (ORC1) in maintaining repressive chromatin structures surrounding the HIV-1 promoter region, thereby contributing to HIV-1 latency. This discovery expands our understanding of the non-replicative functions of the ORC complex and provides a potential therapeutic strategy for HIV-1 cure.

The nucleolar protein GNL3 prevents resection of stalled replication forks.

Faithful DNA replication requires specific proteins that protect replication forks and so prevent the formation of DNA lesions that may damage the genome. Identification of new proteins involved in this process is essential to understand how DNA lesions accumulate in cancer cells and how they tolerate them. Here, we show that human GNL3/nucleostemin, a GTP-binding protein localized mostly in the nucleolus and highly expressed in cancer cells, prevents nuclease-dependent resection of nascent DNA in response to replication stress. We demonstrate that inhibiting origin firing reduces resection. This suggests that the heightened replication origin activation observed upon GNL3 depletion largely drives the observed DNA resection probably due to the exhaustion of the available RPA pool. We show that GNL3 and DNA replication initiation factor ORC2 interact in the nucleolus and that the concentration of GNL3 in the nucleolus is required to limit DNA resection. We propose that the control of origin firing by GNL3 through the sequestration of ORC2 in the nucleolus is critical to prevent nascent DNA resection in response to replication stress.

A miR-361-5p/ ORC6/ PLK1 axis regulates prostate cancer progression.

Prostate cancer (PCa) is the most prevalent malignant tumor of the genitourinary system, and metastatic disease has a significant impact on the prognosis of PCa patients. As a result, knowing the processes of PCa development can help patients achieve better outcomes. Here, we investigated the expression and function of ORC6 in PCa. Our findings indicated that ORC6 was elevated in advanced PCa tissues. Patients with PCa who exhibited high levels of ORC6 had a poor prognosis. Following that, we investigated the function of ORC6 in PCa progression using a variety of functional experiments both in vivo and in vitro, and discovered that ORC6 knockdown inhibited PCa cell proliferation, growth, and migration. Furthermore, RNA-seq was employed to examine the molecular mechanism of PCa progression. The results revealed that ORC6 might promote the expression of PLK1, a serine/threonine kinase in PCa cells. We also discovered that ORC6 as a novel miR-361-5p substrate using database analysis, and miR-361-5p was found to lower ORC6 expression. Additionally, RNA immunoprecipitation (RIP) and luciferase reporter tests revealed that the transcription factor E2F1 could regulate ORC6 expression in PCa cells. PLK1 overexpression or miR-361-5p inhibitor treatment effectively removed the inhibitory effects caused by ORC6 silencing. Notably, our data showed that therapeutically targeting the miR-361-5p/ORC6/PLK1 axis may be a viable therapy option for PCa.

Chromatin Constrains the Initiation and Elongation of DNA Replication.

Eukaryotic chromosomal DNA is faithfully replicated in a complex series of cell-cycle-regulated events that are incompletely understood. Here we report the reconstitution of DNA replication free in solution with purified proteins from the budding yeast Saccharomyces cerevisiae. The system recapitulates regulated bidirectional origin activation; synthesis of leading and lagging strands by the three replicative DNA polymerases Pol α, Pol δ, and Pol ε; and canonical maturation of Okazaki fragments into continuous daughter strands. We uncover a dual regulatory role for chromatin during DNA replication: promoting origin dependence and determining Okazaki fragment length by restricting Pol δ progression. This system thus provides a functional platform for the detailed mechanistic analysis of eukaryotic chromosome replication.

Unique Roles of the Non-identical MCM Subunits in DNA Replication Licensing.

A family of six homologous subunits, Mcm2, -3, -4, -5, -6, and -7, each with its own unique features, forms the catalytic core of the eukaryotic replicative helicase. The necessity of six similar but non-identical subunits has been a mystery since its initial discovery. Recent cryo-EM structures of the Mcm2-7 (MCM) double hexamer, its precursors, and the origin recognition complex (ORC)-Cdc6-Cdt1-Mcm2-7 (OCCM) intermediate showed that each of these subunits plays a distinct role in orchestrating the assembly of the pre-replication complex (pre-RC) by ORC-Cdc6 and Cdt1.

ORChestra coordinates the replication and repair music.

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.

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

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.

Origin recognition complex subunit 6 (ORC6) is a key mediator of LPS-induced NFκB activation and the pro-inflammatory response.

Lipopolysaccharide (LPS)-activated pro-inflammatory responses play a critical role in sepsis, a life-threatening condition. This study investigates the role of origin recognition complex subunit 6 (ORC6) in LPS responses in macrophages and monocytes. Silencing ORC6 using targeted shRNA significantly reduced LPS-induced expression and production of IL-1ß (interleukin-1 beta), TNF-α (tumor necrosis factor alpha), and IL-6 (interleukin-6) in THP-1 human macrophages, peripheral blood mononuclear cells (PBMCs), and bone marrow-derived macrophages (BMDMs). Additionally, ORC6 knockout (KO) via the CRISPR/Cas9 method in THP-1 macrophages inhibited LPS-induced pro-inflammatory responses, while ectopic overexpression of ORC6 enhanced LPS-induced expression and production of pro-inflammatory cytokines. ORC6 is crucial for the activation of the nuclear factor kappa B (NFκB) signaling cascade in macrophages and monocytes. LPS-induced NFκB activation was largely inhibited by ORC6 silencing or KO, but potentiated following ORC6 overexpression. Mechanistically, ORC6 associated with nuclear p65 after LPS stimulation, an interaction necessary for NFκB activation. Overexpression of ORC6 did not recover the reduced pro-inflammatory response to LPS in THP-1 macrophages with silenced p65. Furthermore, the NFκB inhibitor BMS-345,541 nearly eliminated the pro-inflammatory response enhanced by ORC6 overexpression in response to LPS. Further studies revealed that ORC6 depletion inhibited NFκB activation induced by double-stranded RNA (dsRNA) and high mobility group box 1 (HMGB1) in THP-1 macrophages. In vivo experiments demonstrated that macrophage-specific knockdown of ORC6 protected mice from LPS-induced septic shock and inhibited LPS-stimulated production of IL-1ß, TNF-α, and IL-6 in mouse serum. ORC6 silencing also inhibited LPS-induced NFκB activation in ex vivo cultured PBMCs following macrophage-specific knockdown of ORC6. These findings highlight ORC6 as a pivotal mediator in LPS-induced NFκB activation and the pro-inflammatory response in sepsis, suggesting that targeting ORC6 could be a novel therapeutic strategy for managing sepsis and related inflammatory conditions.

DNA replication timing: Biochemical mechanisms and biological significance.

The regulation of DNA replication is a fascinating biological problem both from a mechanistic angle-How is replication timing regulated?-and from an evolutionary one-Why is replication timing regulated? Recent work has provided significant insight into the first question. Detailed biochemical understanding of the mechanism and regulation of replication initiation has made possible robust hypotheses for how replication timing is regulated. Moreover, technical progress, including high-throughput, single-molecule mapping of replication initiation and single-cell assays of replication timing, has allowed for direct testing of these hypotheses in mammalian cells. This work has consolidated the conclusion that differential replication timing is a consequence of the varying probability of replication origin initiation. The second question is more difficult to directly address experimentally. Nonetheless, plausible hypotheses can be made and one-that replication timing contributes to the regulation of chromatin structure-has received new experimental support.

Dynamics of Replication-Associated Protein Levels through the Cell Cycle.

The measurement of dynamic changes in protein level and localization throughout the cell cycle is of major relevance to studies of cellular processes tightly coordinated with the cycle, such as replication, transcription, DNA repair, and checkpoint control. Currently available methods include biochemical assays of cells in bulk following synchronization, which determine protein levels with poor temporal and no spatial resolution. Taking advantage of genetic engineering and live-cell microscopy, we performed time-lapse imaging of cells expressing fluorescently tagged proteins under the control of their endogenous regulatory elements in order to follow their levels throughout the cell cycle. We effectively discern between cell cycle phases and S subphases based on fluorescence intensity and distribution of co-expressed proliferating cell nuclear antigen (PCNA)-mCherry. This allowed us to precisely determine and compare the levels and distribution of multiple replication-associated factors, including Rap1-interacting factor 1 (RIF1), minichromosome maintenance complex component 6 (MCM6), origin recognition complex subunit 1 (ORC1, and Claspin, with high spatiotemporal resolution in HeLa Kyoto cells. Combining these data with available mass spectrometry-based measurements of protein concentrations reveals the changes in the concentration of these proteins throughout the cell cycle. Our approach provides a practical basis for a detailed interrogation of protein dynamics in the context of the cell cycle.

TREX-2-ORC Complex of D. melanogaster Participates in Nuclear Export of Histone mRNA.

The TREX-2-ORC protein complex of D. melanogaster is necessary for the export of the bulk of synthesized poly(A)-containing mRNA molecules from the nucleus to the cytoplasm through the nuclear pores. However, the role of this complex in the export of other types of RNA remains unknown. We have shown that TREX-2-ORC participates in the nuclear export of histone mRNAs: it associates with histone mRNPs, binds to histone H3 mRNA at the 3'-terminal part of the coding region, and participates in the export of histone mRNAs from the nucleus to the cytoplasm.

Replication Licensing Aberrations, Replication Stress, and Genomic Instability.

Strict regulation of DNA replication is of fundamental significance for the maintenance of genome stability. Licensing of origins of DNA replication is a critical event for timely genome duplication. Errors in replication licensing control lead to genomic instability across evolution. Here, we present accumulating evidence that aberrant replication licensing is linked to oncogene-induced replication stress and poses a major threat to genome stability, promoting tumorigenesis. Oncogene activation can lead to defects in where along the genome and when during the cell cycle licensing takes place, resulting in replication stress. We also discuss the potential of replication licensing as a specific target for novel anticancer therapies.

ORIGIN OF RECOGNITION COMPLEX 3 controls the development of maternal excess endosperm in the Paspalum simplex agamic complex (Poaceae).

Pseudogamous apomixis in Paspalum simplex generates seeds with embryos genetically identical to the mother plant and endosperms deviating from the canonical 2(maternal):1(paternal) parental genome contribution into a maternal excess 4m:1p genome ratio. In P. simplex, the gene homologous to that coding for subunit 3 of the ORIGIN OF RECOGNITION COMPLEX (PsORC3) exists in three isogenic forms: PsORC3a is apomixis specific and constitutively expressed in developing endosperm whereas PsORCb and PsORCc are up-regulated in sexual endosperms and silenced in apomictic ones. This raises the question of how the different arrangement and expression profiles of these three ORC3 isogenes are linked to seed development in interploidy crosses generating maternal excess endosperms. We demonstrate that down-regulation of PsORC3b in sexual tetraploid plants is sufficient to restore seed fertility in interploidy 4n×2n crosses and, in turn, its expression level at the transition from proliferating to endoreduplication endosperm developmental stages dictates the fate of these seeds. Furthermore, we show that only when being maternally inherited can PsORC3c up-regulate PsORC3b. Our findings lay the basis for an innovative route-based on ORC3 manipulation-to introgress the apomictic trait into sexual crops and overcome the fertilization barriers in interploidy crosses.

ORC6, a novel prognostic biomarker, correlates with T regulatory cell infiltration in prostate adenocarcinoma: a pan-cancer analysis.

BACKGROUND: The origin recognition complex (ORC), a six-subunit DNA-binding complex, participates in DNA replication in cancer cells. Specifically in prostate cancers, ORC participates the androgen receptor (AR) regulated genomic amplification and tumor proliferation throughout the entire cell cycle. Of note, ORC6, the smallest subunit of ORC, has been reported to be dysregulated in some types of cancers (including prostate cancer), however, its prognostic and immunological significances remain yet to be elucidated. METHODS: In the current study, we comprehensively investigated the potential prognostic and immunological role of ORC6 in 33 human tumors using multiple databases, such as TCGA, Genotype-Tissue Expression, CCLE, UCSC Xena, cBioPortal, Human Protein Atlas, GeneCards, STRING, MSigDB, TISIDB, and TIMER2 databases. RESULTS: ORC6 expression was significantly upregulated in 29 types of cancers compared to the corresponding normal adjacent tissues. ORC6 overexpression correlated with higher stage and worse prognostic outcomes in most cancer types analyzed. Additionally, ORC6 was involved in the cell cycle pathway, DNA replication, and mismatch repair pathways in most tumor types. A negative correlation was observed between the tumor endothelial cell infiltration and ORC6 expression in almost all tumors, whereas the immune infiltration of T regulatory cell was noted to be statistically positively correlated with the expression of ORC6 in prostate cancer tissues. Furthermore, in most tumor types, immunosuppression-related genes, especially TGFBR1 and PD-L1 (CD274), exhibited a specific correlation with the expression of ORC6. CONCLUSIONS: This comprehensive pan-cancer analysis revealed that ORC6 expression serves as a prognostic biomarker and that ORC6 is involved in the regulation of various biological pathways, the tumor microenvironment, and the immunosuppression status in several human cancers, suggesting its potential diagnostic, prognostic, and therapeutic value in pan-cancer, especially in prostate adenocarcinoma.