The initiation of DNA replication in eukaryotic cells begins with the assembly of pre-replicative complexes (pre-RCs) at many sites along each chromosome during the G1 phase of the cell cycle. Pre-RCs license each chromosome for duplication during S phase and mark the origins of DNA replication. In this Review, we discuss and contextualize recent findings identifying the mechanisms of origin recognition and pre-RC assembly mediated by the origin recognition complex (ORC), Cdc6 and the Mcm2-Mcm7 (Mcm2-7) hexamer bound to Cdt1. We also present comprehensive videos that demonstrate the multiple mechanisms for pre-RC assembly and compare the structures of the complexes involved in human and Saccharomyces cerevisiae cells.

Eukaryotic DNA replication initiates from genomic loci known as origins. At budding yeast origins like ARS1, a double hexamer (DH) of the MCM replicative helicase is assembled by origin recognition complex (ORC), Cdc6 and Cdt1 by sequential hexamer loading from two opposed ORC binding sites. Cyclin-dependent kinase (CDK) inhibits DH assembly, which prevents re-replication by restricting helicase loading to the G1 phase. Here, we show that an intrinsically disordered region (IDR) in the Orc2 subunit promotes interaction between ORC and the first loaded, closed-ring MCM hexamer (the MCM-ORC (MO) intermediate). CDK-dependent phosphorylation of this IDR blocks MO formation and DH assembly. We show that MO stabilizes ORC at lower-affinity binding sites required for second hexamer loading. Origins comprising two high-affinity ORC sites can assemble DH efficiently without MO by independently loading single hexamers. Strikingly, these origins escape CDK inhibition in vitro and in vivo. Our work reveals mechanistic plasticity in MCM loading with implications for understanding how CDK regulation has shaped yeast origin evolution and how natural, strong origins might escape cell cycle regulation. We also identify key steps common to loading pathways, with implications for understanding how MCM is loaded in other eukaryotes.

Following eukaryotic genome replication, the ring-shaped cohesin complex embraces the two newly synthesized sister chromatids, enabling their faithful segregation during cell divisions. Replisome passage through cohesin rings has been envisioned as a fail-safe mechanism that ensures co-entrapment of replication products—whether replisomes can indeed pass through cohesin rings remains unknown. Here, we use biochemical reconstitution and single-molecule fluorescence microscopy to directly visualize replisome-cohesin encounters. We find that the translocating eukaryotic replicative Cdc45-Mcm2-7-GINS (CMG) helicase, unlike other obstacles of similar size, readily passes through cohesin rings. Fully reconstituted replisomes also pass cohesin rings to leave both replication products trapped inside. Replisome passage is primarily aided by DNA polymerases α and ε, a finding that necessitates re-evaluation of canonical cohesion establishment factor roles. Our findings demonstrate the existence of a simple mechanism that links genome replication with chromosome segregation: replisome passage through cohesin rings.

The DNA replication checkpoint is crucial for maintaining genome stability after genotoxic stress; without it, stalled DNA replication forks cannot restart normally, excess DNA replication origins are activated, DNA damage and single-stranded DNA (ssDNA) accumulate, S phase does not finish, and cells die. Preventing excess origin firing suppresses all these effects. Here, we show in human cells that when replication is not restrained by a functional checkpoint, excess DNA synthesis sequesters the processivity factor PCNA and its loader, replication factor C (RFC), preventing normal fork restart. Nascent DNA ends unprotected by RFC/PCNA are attacked by the helicase-like transcription factor (HLTF), causing irreversible replication fork collapse and hyperaccumulation of ssDNA. This explains how the checkpoint stabilizes stalled replication forks and has implications for how origin firing is normally coordinated with fork progression. Loss of HLTF suppresses fork collapse and cell lethality in checkpoint-deficient cells, which has implications for how resistance to anti-checkpoint therapies may arise.

Aneuploidy, characterised by an imbalanced chromosome copy number, is the cause of chromosomal disorders[1][1] as well as a hallmark of cancer[2][2]. Beyond pathological conditions, recent studies highlight the presence of lineage-specific recurrent aneuploidies in normal tissues[3][3]–[5][4]. Despite its prevalence, the functional significance of aneuploidies in cellular dysfunction and diseases remains elusive, hindered by experimental challenges in engineering relevant aneuploidy models for mechanistic investigation. Here, we discover that the centromeric recruitment of catalytically dead Cas9 (dCas9) induces efficient chromosome-specific mis-segregation, applicable to all 24 human chromosomes. We show that the mis-segregation phenotype is driven by dCas9-induced kinetochore chromatin relaxation, resulting in whole chromosome aneuploidy or arm-level aneuploidy with centromeric breakage. In induced pluripotent stem cells, we demonstrate aneuploidy manipulation for chromosome 13, 21, X, and Y. In primary renal epithelial cells, we engineered chromosome 3(p) loss, the tumour-initiating event in clear cell renal cell carcinoma (ccRCC)[6][5].. Overall, we describe a simple, efficient, and versatile approach for chromosome-specific aneuploidy generation that can facilitate preclinical aneuploid model development for functional interrogation of aneuploidy. ### Competing Interest Statement S.T. has received speaking fees from Roche, Astra Zeneca, Novartis, and Ipsen. S.T. has the following patents filed: Indel mutations as a therapeutic target and predictive biomarker PCTGB2018/051892, PCTGB2018/051893, and P113326GB. The Francis Crick Institute, https://ror.org/04tnbqb63, Cancer Research UK, , C50947/A29911 VHL Alliance, , US Department of Defense, , W81XWH2210764 Beijing Natural Science Foundation, , 7242083 [1]: #ref-1 [2]: #ref-2 [3]: #ref-3 [4]: #ref-5 [5]: #ref-6

Replication stress, a hallmark of cancer cells, is detected by checkpoint mechanisms that trigger a range of cellular responses. Among these, the preservation of replication fork integrity is crucial for ensuring survival in the presence of DNA damage. In budding yeast checkpoint mutants, DNA damage leads to irreversible replication fork arrest and subsequent cell death, though the underlying mechanism remains unclear. Our study reveals that several DNA processing factors, including Rad51, the Rad5 HIRAN and helicase domains, and the catalytic activity of Mus81, contribute to this lethality. Nevertheless, their roles are masked by their essential functions in cell survival after damage removal. Additionally, we show that these factors, along with Exo1, drive the gradual degradation of nascent DNA at replication forks upon DNA damage. Notably, this degradation can be mitigated by expression of human PrimPol, which is absent in yeast. These findings suggest that the essential role of S-phase checkpoints upon DNA damage is to safeguard stalled replication forks from aberrant processing, thereby preserving nascent DNA integrity.

Understanding how DNA replication forks stall and restart and how the DNA replication checkpoint prevents irreversible fork collapse in molecular detail are crucial for understanding how cells maintain stable genomes and how they prevent the genetic instability that drives cancer. Here, we describe the reconstitution of fork stalling and restart with purified budding yeast proteins. After nucleotide depletion, leading-strand DNA synthesis quickly stops but CMG helicase continues to unwind, and Okazaki fragments continue to initiate on the lagging strand. Incomplete Okazaki fragments sequester PCNA, RFC, and DNA polymerases δ and ε, which prevents normal DNA synthesis restart and exposes nascent DNA to nuclease attack. The DNA replication checkpoint restrains fork progression, which limits this sequestration, protecting stalled forks from collapse and ensuring restart.

The DNA damage checkpoint is crucial for maintaining genome stability after genotoxic stress; without it, excess DNA replication origins are activated, stalled DNA replication forks cannot restart normally, high levels of DNA damage and single-stranded DNA (ssDNA) accumulate and cells cannot complete S phase. Preventing excess origin firing suppresses all these effects. Here we show that when replication is not restrained by a functional checkpoint, excess DNA synthesis sequesters the processivity factor PCNA and its loader RFC, preventing normal fork restart. Nascent DNA ends unprotected by RFC/PCNA are attacked by the Helicase-Like Transcription Factor (HLTF), causing irreversible replication fork collapse and hyperaccumulation of single-stranded DNA. This explains how the checkpoint stabilizes stalled replication forks and has implications for how origin firing is normally coordinated with fork progression. Loss of HLTF suppresses fork collapse and cell lethality in checkpoint-deficient cells, which has implications for how resistance to anti-checkpoint therapies may arise. ### Competing Interest Statement The authors have declared no competing interest.

Eukaryotes have one replicative helicase known as CMG, which centrally organizes and drives the replisome, and leads the way at the front of replication forks. Obtaining a deep mechanistic understanding of the dynamics of CMG is critical to elucidating how cells achieve the enormous task of efficiently and accurately replicating their entire genome once per cell cycle. Single-molecule techniques are uniquely suited to quantify the dynamics of CMG due to their unparalleled temporal and spatial resolution. Nevertheless, single-molecule studies of CMG motion have thus far relied on pre-formed CMG purified from cells as a complex, which precludes the study of the steps leading up to its activation. Here, we describe a hybrid ensemble and single-molecule assay that allowed imaging at the single-molecule level of the motion of fluorescently labeled CMG after fully reconstituting its assembly and activation from 36 different purified S. cerevisiae polypeptides. This assay relies on the double functionalization of the ends of a linear DNA substrate with two orthogonal attachment moieties, and can be adapted to study similarly complex DNA-processing mechanisms at the single-molecule level.

Understanding how DNA replication forks stall and restart and how the DNA damage checkpoint prevents irreversible fork collapse in molecular detail are crucial for understanding how cells maintain stable genomes and how they prevent the genetic instability that drives cancer. Here we describe the reconstitution of fork stalling and restart with purified budding yeast proteins. After nucleotide depletion, leading strand DNA synthesis quickly stops but CMG helicase continues to unwind and Okazaki fragments continue to initiate on the lagging strand. Incomplete Okazaki fragments sequester PCNA, RFC and DNA polymerases δ and ε which prevents normal DNA synthesis restart and exposes nascent DNA to nuclease attack. The DNA damage checkpoint limits this sequestration by restraining fork progression, which protects stalled forks from collapse and ensures restart. ### Competing Interest Statement The authors have declared no competing interest.

To prevent detrimental chromosome re-replication, DNA loading of a double hexamer of the minichromosome maintenance (MCM) replicative helicase is temporally separated from DNA unwinding. Upon S-phase transition in yeast, DNA unwinding is achieved in two steps: limited opening of the double helix and topological separation of the two DNA strands. First, Cdc45, GINS and Polε engage MCM to assemble a double CMGE with two partially separated hexamers that nucleate DNA melting. In the second step, triggered by Mcm10, two CMGEs separate completely, eject the lagging-strand template and cross paths. To understand Mcm10 during helicase activation, we used biochemical reconstitution with cryogenic electron microscopy. We found that Mcm10 splits the double CMGE by engaging the N-terminal homo-dimerization face of MCM. To eject the lagging strand, DNA unwinding is started from the N-terminal side of MCM while the hexamer channel becomes too narrow to harbor duplex DNA.

Eukaryotic DNA replication begins with the loading of the MCM replicative DNA helicase as a head-to-head double hexamer at origins of DNA replication(1-3). Our current understanding of how the double hexamer is assembled by the origin recognition complex (ORC), CDC6 and CDT1 comes mostly from budding yeast. Here we characterize human double hexamer (hDH) loading using biochemical reconstitution and cryo-electron microscopy with purified proteins. We show that the human double hexamer engages DNA differently from the yeast double hexamer (yDH), and generates approximately five base pairs of underwound DNA at the interface between hexamers, as seen in hDH isolated from cells(4). We identify several differences from the yeast double hexamer in the order of factor recruitment and dependencies during hDH assembly. Unlike in yeast(5-8), the ORC6 subunit of the ORC is not essential for initial MCM recruitment or hDH loading, but contributes to an alternative hDH assembly pathway that requires an intrinsically disordered region in ORC1, which may work through a MCM-ORC intermediate. Our work presents a detailed view of how double hexamers are assembled in an organism that uses sequence-independent replication origins, provides further evidence for diversity in eukaryotic double hexamer assembly mechanisms(9), and represents a first step towards reconstitution of DNA replication initiation with purified human proteins.

Summary Eukaryotic DNA replication initiates from multiple genomic loci known as origins. At budding yeast origins like ARS1, a double hexamer (DH) of the MCM replicative helicase is assembled by Origin Recognition Complex (ORC), Cdc6 and Cdt1 via sequential hexamer loading from two opposed ORC binding sites. Cyclin Dependent Kinase (CDK) inhibits DH assembly, which prevents re-replication by restricting helicase loading to G1 phase. Here we show that an intrinsically disordered region (IDR) in the Orc2 subunit promotes interaction between ORC and the first loaded, closed-ring MCM hexamer (the MO intermediate); CDK phosphorylation of this IDR blocks MO formation and DH assembly. We show that MO functions by stabilising ORC at the lower affinity binding sites required for second hexamer loading. Origins comprising two high affinity ORC sites can assemble DH efficiently without MO by independently loading single hexamers; these origins escape CDK inhibition in vitro and in vivo . Our work reveals mechanistic plasticity in MCM loading with implications for understanding how CDK regulation has shaped yeast origin evolution and how natural origins might escape cell cycle regulation. We also identify key steps common to loading pathways, with implications for understanding how MCM is loaded in other eukaryotes.

The eukaryotic replicative helicase CMG centrally orchestrates the replisome and leads the way at the front of replication forks. Understanding the motion of CMG on the DNA is therefore key to our understanding of DNA replication. In vivo, CMG is assembled and activated through a cell-cycle-regulated mechanism involving 36 polypeptides that has been reconstituted from purified proteins in ensemble biochemical studies. Conversely, single-molecule studies of CMG motion have thus far relied on pre-formed CMG assembled through an unknown mechanism upon overexpression of individual constituents. Here, we report the activation of CMG fully reconstituted from purified yeast proteins and the quantification of its motion at the single-molecule level. We observe that CMG can move on DNA in two ways: by unidirectional translocation and by diffusion. We demonstrate that CMG preferentially exhibits unidirectional translocation in the presence of ATP, whereas it preferentially exhibits diffusive motion in the absence of ATP. We also demonstrate that nucleotide binding halts diffusive CMG independently of DNA melting. Taken together, our findings support a mechanism by which nucleotide binding allows newly assembled CMG to engage with the DNA within its central channel, halting its diffusion and facilitating the initial DNA melting required to initiate DNA replication.

Whole-genome duplication (WGD) is a frequent event in cancer evolution and an important driver of aneuploidy. The role of the p53 tumor suppressor in WGD has been enigmatic: p53 can block the proliferation of tetraploid cells, acting as a barrier to WGD, but can also promote mitotic bypass, a key step in WGD via endoreduplication. In wild-type (WT) p53 tumors, WGD is frequently associated with activation of the E2F pathway, especially amplification of CCNE1, encoding cyclin E1. Here, we show that elevated cyclin E1 expression causes replicative stress, which activates ATR- and Chk1-dependent G2 phase arrest. p53, via its downstream target p21, together with Wee1, then inhibits mitotic cyclin-dependent kinase activity sufficiently to activate APC/CCdh1 and promote mitotic bypass. Cyclin E expression suppresses p53-dependent senescence after mitotic bypass, allowing cells to complete endoreduplication. Our results indicate that p53 can contribute to cancer evolution through the promotion of WGD.

Concomitant with DNA replication, the chromosomal cohesin complex establishes cohesion between newly replicated sister chromatids. Cohesion establishment requires acetylation of conserved cohesin lysine residues by Eco1 acetyltransferase. Here, we explore how cohesin acetylation is linked to DNA replication. Biochemical reconstitution of replication-coupled cohesin acetylation reveals that transient DNA structures, which form during DNA replication, control the acetylation reaction. As polymerases complete lagging strand replication, strand displacement synthesis produces DNA flaps that are trimmed to result in nicked double-stranded DNA. Both flaps and nicks stimulate cohesin acetylation, while subsequent nick ligation to complete Okazaki fragment maturation terminates the acetylation reaction. A flapped or nicked DNA substrate constitutes a transient molecular clue that directs cohesin acetylation to a window behind the replication fork, next to where cohesin likely entraps both sister chromatids. Our results provide an explanation for how DNA replication is linked to sister chromatid cohesion establishment.

Abstract Chromatin replication involves the assembly and activity of the replisome within the nucleosomal landscape. At the core of the replisome is the Mcm2-7 complex (MCM), which is loaded onto DNA after binding to the Origin Recognition Complex (ORC). In yeast, ORC is a dynamic protein that diffuses rapidly along DNA, unless halted by origin recognition sequences. However, less is known about the dynamics of ORC proteins in the presence of nucleosomes and attendant consequences for MCM loading. To address this, we harnessed an in vitro single-molecule approach to interrogate a chromatinized origin of replication. We find that ORC binds the origin of replication with similar efficiency independently of whether the origin is chromatinized, despite ORC mobility being reduced by the presence of nucleosomes. Recruitment of MCM also proceeds efficiently on a chromatinized origin, but subsequent movement of MCM away from the origin is severely constrained. These findings suggest that chromatinized origins in yeast are essential for the local retention of MCM, which may facilitate subsequent assembly of the replisome.