To prevent rereplication of genomic segments, the eukaryotic cell cycle is divided into two nonoverlapping phases. During late mitosis and G1 replication origins are "licensed" by loading MCM2-7 double hexamers and during S phase licensed replication origins activate to initiate bidirectional replication forks. Replication forks can stall irreversibly, and if two converging forks stall with no intervening licensed origin-a "double fork stall" (DFS)-replication cannot be completed by conventional means. We previously showed how the distribution of replication origins in yeasts promotes complete genome replication even in the presence of irreversible fork stalling. This analysis predicts that DFSs are rare in yeasts but highly likely in large mammalian genomes. Here we show that complementary strand synthesis in early mitosis, ultrafine anaphase bridges, and G1-specific p53-binding protein 1 (53BP1) nuclear bodies provide a mechanism for resolving unreplicated DNA at DFSs in human cells. When origin number was experimentally altered, the number of these structures closely agreed with theoretical predictions of DFSs. The 53BP1 is preferentially bound to larger replicons, where the probability of DFSs is higher. Loss of 53BP1 caused hypersensitivity to licensing inhibition when replication origins were removed. These results provide a striking convergence of experimental and theoretical evidence that unreplicated DNA can pass through mitosis for resolution in the following cell cycle.uring the eukaryotic cell cycle, the genome must be precisely duplicated with no sections left unreplicated and no sections replicated more than once. To prevent rereplication, the process is divided into two nonoverlapping phases: during late mitosis and G1 replication origins are "licensed" for subsequent use by loading MCM2-7 double hexamers, and during S phase DNAbound MCM2-7 is activated to form processive CMG (CDC45-MCM-GINS) helicases that drive replication fork progression. The prohibition of origin licensing during S phase and G2 ensures that rereplication of DNA cannot occur. However, the inability to license new origins after the onset of S phase provides a challenge for the cell to fully replicate the genome using its finite supply of licensed origins. Replication forks can irreversibly stall when they encounter unusual structures on the DNA, such as DNA damage or tightly bound protein-DNA complexes.When replication initiation occurs at a licensed replication origin the MCM2-7 double hexamer forms a pair of bidirectionally orientated CMG helicases (1-3). If one fork irreversibly stalls, the converging fork from a neighboring origin can compensate by replicating all of the DNA up to the stalled fork. However, if two converging forks both stall and there is no licensed origin between them-a "double fork stall" (DFS)-new replicative machinery cannot be recruited to replicate the intervening DNA (4). To compensate for this potential for underreplication, origins are licensed redundantly, with most (typically >70%) remaining dorm...

During S phase, the entire genome must be precisely duplicated, with no sections of DNA left unreplicated. Here, we develop a simple mathematical model to describe the probability of replication failing due to the irreversible stalling of replication forks. We show that the probability of complete genome replication is maximized if replication origins are evenly spaced, the largest inter-origin distances are minimized, and the end-most origins are positioned close to chromosome ends. We show that origin positions in the yeast Saccharomyces cerevisiae genome conform to all three predictions thereby maximizing the probability of complete replication if replication forks stall. Origin positions in four other yeasts—Kluyveromyces lactis, Lachancea kluyveri, Lachancea waltii and Schizosaccharomyces pombe—also conform to these predictions. Equating failure rates at chromosome ends with those in chromosome interiors gives a mean per nucleotide fork stall rate of ∼5 × 10−8, which is consistent with experimental estimates. Using this value in our theoretical predictions gives replication failure rates that are consistent with data from replication origin knockout experiments. Our theory also predicts that significantly larger genomes, such as those of mammals, will experience a much greater probability of replication failure genome-wide, and therefore will likely require additional compensatory mechanisms.

The replication of DNA is initiated at particular sites on the genome called replication origins (ROs). Understanding the constraints that regulate the distribution of ROs across different organisms is fundamental for quantifying the degree of replication errors and their downstream consequences. Using a simple probabilistic model, we generate a set of predictions on the extreme sensitivity of error rates to the distribution of ROs, and how this distribution must therefore be tuned for genomes of vastly different sizes. As genome size changes from megabases to gigabases, we predict that regularity of RO spacing is lost, that large gaps between ROs dominate error rates but are heavily constrained by the mean stalling distance of replication forks, and that, for genomes spanning ∼100 megabases to ∼10 gigabases, errors become increasingly inevitable but their number remains very small (three or less). Our theory predicts that the number of errors becomes significantly higher for genome sizes greater than ∼10 gigabases. We test these predictions against datasets in yeast, Arabidopsis, Drosophila, and human, and also through direct experimentation on two different human cell lines. Agreement of theoretical predictions with experiment and datasets is found in all cases, resulting in a picture of great simplicity, whereby the density and positioning of ROs explain the replication error rates for the entire range of eukaryotes for which data are available. The theory highlights three domains of error rates: negligible (yeast), tolerable (metazoan), and high (some plants), with the human genome at the extreme end of the middle domain.T he proper maintenance of genetic information is of fundamental importance to the survival of all organisms, and many molecular mechanisms exist to ensure that the genetic sequence encoded by DNA is maintained unaltered generation after generation (1-3). To preserve the integrity of genetic information and to avoid aberrant ploidy, it is crucial that the entire DNA is copied exactly once; replicating only part of the DNA results in potential corruption of genes, and replicating certain parts of the DNA more than once would perturb chromosome structure and strongly affect gene dosage (4-6). Not surprisingly, regions of underreplicated and overreplicated DNA are common in cancer (7,8).DNA replication is a particularly complex process in eukaryotic organisms with large genomes distributed across multiple chromosomes. Multiple checkpoints exist to ensure that, once replication starts, the whole DNA is faithfully replicated before the chromosomes are segregated. Underreplication and overreplication of DNA are prevented by using predefined points of replication initiation called replication origins (ROs) (3, 9).During late mitosis and the G1 phase of the cell division cycle, each potential RO is "licensed" for a single initiation event by being loaded with minichromosome maintenance proteins 2-7 (MCM2-7) double hexamers. To prevent rereplication of DNA segments, the ability to license new origins...

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