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Minichromosome maintenance proteins (MCMs) are DNA-dependent ATPases that bind to replication origins and license them to support a single round of DNA replication. A large excess of MCM2–7 assembles on chromatin in G1 phase as pre-replication complexes (pre-RCs), of which only a fraction become the productive CDC45–MCM–GINS (CMG) helicases that are required for genome duplication 1 – 4 . It remains unclear why cells generate this surplus of MCMs, how they manage to sustain it across multiple generations, and why even a mild reduction in the MCM pool compromises the integrity of replicating genomes 5 , 6 . Here we show that, for daughter cells to sustain error-free DNA replication, their mother cells build up a nuclear pool of MCMs both by recycling chromatin-bound (parental) MCMs and by synthesizing new (nascent) MCMs. Although all MCMs can form pre-RCs, it is the parental pool that is inherently stable and preferentially matures into CMGs. By contrast, nascent MCM3–7 (but not MCM2) undergo rapid proteolysis in the cytoplasm, and their stabilization and nuclear translocation require interaction with minichromosome-maintenance complex-binding protein (MCMBP), a distant MCM paralogue 7 , 8 . By chaperoning nascent MCMs, MCMBP safeguards replicating genomes by increasing chromatin coverage with pre-RCs that do not participate on replication origins but adjust the pace of replisome movement to minimize errors during DNA replication. Consequently, although the paucity of pre-RCs in MCMBP-deficient cells does not alter DNA synthesis overall, it increases the speed and asymmetry of individual replisomes, which leads to DNA damage. The surplus of MCMs therefore increases the robustness of genome duplication by restraining the speed at which eukaryotic cells replicate their DNA. Alterations in physiological fork speed might thus explain why even a minor reduction in MCM levels destabilizes the genome and predisposes to increased incidence of tumour formation. Mother cells recycle parental MCMs and simultaneously synthesize nascent MCMs, both of which are inherited by daughter cells, in which the former are preferentially used to form active replisomes and the latter adjust the pace of replisome movement to minimize errors during DNA replication.

The entry of mammalian cells into the DNA synthesis phase (S phase) represents a key event in cell division 1 . According to current models of the cell cycle, the kinase CDC7 constitutes an essential and rate-limiting trigger of DNA replication, acting together with the cyclin-dependent kinase CDK2. Here we show that CDC7 is dispensable for cell division of many different cell types, as determined using chemical genetic systems that enable acute shutdown of CDC7 in cultured cells and in live mice. We demonstrate that another cell cycle kinase, CDK1, is also active during G1/S transition both in cycling cells and in cells exiting quiescence. We show that CDC7 and CDK1 perform functionally redundant roles during G1/S transition, and at least one of these kinases must be present to allow S-phase entry. These observations revise our understanding of cell cycle progression by demonstrating that CDK1 physiologically regulates two distinct transitions during cell division cycle, whereas CDC7 has a redundant function in DNA replication. In addition to CDC7, the cell cycle kinase CDK1 has a pivotal role in the G1/S transition of cells, a finding that revises our current understanding of cell cycle progression.

This Opinion article discusses recent studies that have provided new insights into the mechanisms of common fragile site instability and the resulting genomic effects, which include the generation of focal copy number alterations that affect the genomic landscape of many cancers. Ever since initial suggestions that instability at common fragile sites (CFSs) could be responsible for chromosome rearrangements in cancers, CFSs and associated genes have been the subject of numerous studies, leading to questions and controversies about their role and importance in cancer. It is now clear that CFSs are not frequently involved in translocations or other cancer-associated recurrent gross chromosome rearrangements. However, recent studies have provided new insights into the mechanisms of CFS instability, their effect on genome instability, and their role in generating focal copy number alterations that affect the genomic landscape of many cancers.

Defects in DNA replication, or in the pathways evolved to correct DNA replication problems, can cause genomic instability and disease. Zeman and Cimprich discuss recent advances in our understanding of the cellular responses to replication stress. Replication stress is a complex phenomenon that has serious implications for genome stability, cell survival and human disease. Generation of aberrant replication fork structures containing single-stranded DNA activates the replication stress response, primarily mediated by the kinase ATR (ATM- and Rad3-related). Along with its downstream effectors, ATR stabilizes and helps to restart stalled replication forks, avoiding the generation of DNA damage and genome instability. Understanding this response may be key to diagnosing and treating human diseases caused by defective responses to replication stress.

Genome duplication is safeguarded by constantly adjusting the activity of the replicative CMG (CDC45-MCM2-7-GINS) helicase. However, minichromosome maintenance proteins (MCMs)—the structural core of the CMG helicase—have never been visualized at sites of DNA synthesis inside a cell (the so-called MCM paradox). Here, we solve this conundrum by showing that anti-MCM antibodies primarily detect inactive MCMs. Upon conversion of inactive MCMs to CMGs, factors that are required for replisome activity bind to the MCM scaffold and block MCM antibody binding sites. Tagging of endogenous MCMs by CRISPR-Cas9 bypasses this steric hindrance and enables MCM visualization at active replisomes. Thus, by defining conditions for detecting the structural core of the replicative CMG helicase, our results explain the MCM paradox, provide visual proof that MCMs are an integral part of active replisomes in vivo, and enable the investigation of replication dynamics in living cells exposed to a constantly changing environment. For several decades the MCM2-7 proteins, the core of the DNA replicative helicase, eluded detection at DNA replication sites. Here, the authors solve this conundrum by gene editing, which enables visualization of replication dynamics in living cells.

Bacterial chromosomes are folded to compact DNA and facilitate cellular processes. Studying model bacteria has revealed aspects of chromosome folding that are applicable to many species. Primarily controlled by nucleoid-associated proteins, chromosome folding is hierarchical, from large-scale macrodomains to smaller-scale structures that influence DNA transactions, including replication and transcription. Here we review the environmentally regulated, architectural and regulatory roles of nucleoid-associated proteins and the implications for bacterial cell biology. We also highlight similarities and differences in the chromosome folding mechanisms of bacteria and eukaryotes.Advances in sequencing- and imaging-based techniques for chromosome structure analysis have led to a mature understanding of bacterial chromosome structure and dynamics. In this Review, Dame, Rashid and Grainger discuss the hierarchical nature of bacterial chromosome structure and how it is influenced by diverse types of nucleoid-associated proteins. Furthermore, they describe roles for nucleoid-associated proteins and chromosome structure, including in gene expression, chromosome segregation and cell cycle regulation.

The 3D organization of mammalian chromatin was described more than 30 years ago by visualizing sites of DNA synthesis at different times during the S phase of the cell cycle. These early cytogenetic studies revealed structurally stable chromosome domains organized into subnuclear compartments. Active-gene-rich domains in the nuclear interior replicate early, whereas more condensed chromatin domains that are largely at the nuclear and nucleolar periphery replicate later. During the past decade, this spatiotemporal DNA replication programme has been mapped along the genome and found to correlate with epigenetic marks, transcriptional activity and features of 3D genome architecture such as chromosome compartments and topologically associated domains. But the causal relationship between these features and DNA replication timing and the regulatory mechanisms involved have remained an enigma. The recent identification of cis-acting elements regulating the replication time and 3D architecture of individual replication domains and of long non-coding RNAs that coordinate whole chromosome replication provide insights into such mechanisms.

SAMHD1 was previously characterized as a dNTPase that protects cells from viral infections. Mutations in SAMHD1 are implicated in cancer development and in a severe congenital inflammatory disease known as Aicardi–Goutières syndrome. The mechanism by which SAMHD1 protects against cancer and chronic inflammation is unknown. Here we show that SAMHD1 promotes degradation of nascent DNA at stalled replication forks in human cell lines by stimulating the exonuclease activity of MRE11. This function activates the ATR–CHK1 checkpoint and allows the forks to restart replication. In SAMHD1-depleted cells, single-stranded DNA fragments are released from stalled forks and accumulate in the cytosol, where they activate the cGAS–STING pathway to induce expression of pro-inflammatory type I interferons. SAMHD1 is thus an important player in the replication stress response, which prevents chronic inflammation by limiting the release of single-stranded DNA from stalled replication forks. SAMHD1 has an essential role in the replication stress response and prevents inflammation by activating the MRE11 nuclease to degrade nascent DNA strands at stalled replication forks, thus enabling replication.

Common fragile sites (CFSs) are difficult-to-replicate regions of eukaryotic genomes that are sensitive to replication stress and that require resolution by the MUS81–EME1 endonuclease to re-initiate POLD3-dependent DNA synthesis in early mitosis; this study defines the specific pathway of events causing the CFS fragility phenotype. Inbuilt chromosome fragility Common fragile sites (CFSs) are regions of eukaryotic genomes that are sensitive to DNA replication stressors. They are stably maintained and replicated in the human genome most of the time, but have been associated with some cancers and genetic disease. CFS 'expression' was shown recently to be a programmed event promoted by the MUS81 endonuclease, rather than disruption of the chromosome structure caused by mechanical forces in mitosis. Ian Hickson and colleagues have now defined the specific chain of events causing CFSs. They describe a pathway of unscheduled DNA synthesis regulated by the MUS81 endonuclease, the POLD3 subunit of DNA polymerase delta and the prophase pathway in early mitosis. Oncogene-induced DNA replication stress has been implicated as a driver of tumorigenesis 1 . Many chromosomal rearrangements characteristic of human cancers originate from specific regions of the genome called common fragile sites (CFSs) 2 , 3 , 4 , 5 . CFSs are difficult-to-replicate loci that manifest as gaps or breaks on metaphase chromosomes (termed CFS ‘expression’), particularly when cells have been exposed to replicative stress 6 . The MUS81–EME1 structure-specific endonuclease promotes the appearance of chromosome gaps or breaks at CFSs following replicative stress 7 , 8 , 9 . Here we show that entry of cells into mitotic prophase triggers the recruitment of MUS81 to CFSs. The nuclease activity of MUS81 then promotes POLD3-dependent DNA synthesis at CFSs, which serves to minimize chromosome mis-segregation and non-disjunction. We propose that the attempted condensation of incompletely duplicated loci in early mitosis serves as the trigger for completion of DNA replication at CFS loci in human cells. Given that this POLD3-dependent mitotic DNA synthesis is enhanced in aneuploid cancer cells that exhibit intrinsically high levels of chromosomal instability (CIN + ) and replicative stress, we suggest that targeting this pathway could represent a new therapeutic approach.

The nucleolus is the major site for synthesis of ribosomes, complex molecular machines that are responsible for protein synthesis. A wealth of research over the past 20 years has clearly indicated that both quantitative and qualitative alterations in ribosome biogenesis can drive the malignant phenotype via dysregulation of protein synthesis. However, numerous recent proteomic, genomic, and functional studies have implicated the nucleolus in the regulation of processes that are unrelated to ribosome biogenesis, including DNA-damage response, maintenance of genome stability and its spatial organization, epigenetic regulation, cell-cycle control, stress responses, senescence, global gene expression, as well as assembly or maturation of various ribonucleoprotein particles. In this review, the focus will be on features of rDNA genes, which make them highly vulnerable to DNA damage and intra- and interchromosomal recombination as well as built-in mechanisms that prevent and repair rDNA damage, and how dysregulation of this interplay affects genome-wide DNA stability, gene expression and the balance between euchromatin and heterochromatin. We will also present the most recent insights into how malfunction of these cellular processes may be a central driving force of human malignancies, and propose a promising new therapeutic approach for the treatment of cancer.