Unreplicated DNA remaining from unperturbed S phases passes through mitosis for resolution in daughter cells

Abstract

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...

Inevitability and containment of replication errors for eukaryotic genome lengths spanning megabase to gigabase

“…As such, large regions of repetitive DNA will not be sequenced accurately and yet are likely to contain ROs. These false negatives imply that the largest replicons measured provide an upper bound rather than a definite value, although we do not expect large numbers of missed ROs (19). The future use of more advanced techniques, for example, single-cell sequencing, will shed more light on this aspect.…”

“…The probability of DFSs is very small in yeasts due to the small genome size, and optimization of the RO positions by lowering R reduces this even further. However, as discussed above, organisms with larger genomes have a significantly higher probability of DFS events, which results in the need for additional molecular mechanisms to cope with the consequences (19), and the presence of such mechanisms means there is little to be gained in uniformly ordering ROs on the genomes. Thus, our expectation is that R should be significantly larger in organisms with larger genomes compared with the values found in yeast.…”

“…For organisms with larger genomes, such as typical vertebrates and plants, DFSs are highly likely, even if the mean replicon length is small. These organisms therefore require mechanisms to deal with DFSs, and, in related experimental work, we provide experimental evidence for one such postreplicative mechanism (19). For cells with such repair mechanisms, the burden of equally spacing ROs is lifted; far more important is the distribution of larger replicons (relative to N s ) from which DFS events are most likely to arise.…”

SIR2 suppresses replication gaps and genome instability by balancing replication between repetitive and unique sequences

“…Consistent with our results, a report from Moreno et al (20) emphasizes the importance of the interorigin spacing in generation of replication gaps: These investigators identified regions of late replication by the presence of ultrafine anaphase bridges. They showed that formation of such bridges was inversely correlated with the density of licensed origins, and, furthermore, that the numbers of bridges increased when the Mcm5 helicase, which is required for origin licensing, was targeted using RNAi (20). More work will be required to make more-specific genome-wide predictions of regions expected to be susceptible to incomplete replication.…”

Knotty Problems during Mitosis: Mechanistic Insight into the Processing of Ultrafine DNA Bridges in Anaphase

“…According to recent modeling studies, cells with large genomes enter mitosis with, on average, three underreplicated sites per cell, even in unperturbed growth conditions. This problem is exacerbated by conditions that induce replication stress or by reducing the number of origins Moreno et al 2016).…”