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

Abstract

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...

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

“…This experimental work strongly supports the theoretical analysis of DFSs in organisms of differing genome size that we present in an accompanying paper (10).…”

“…This mechanism depends on the resolution and segregation of topologically intertwined strands and could require a limited amount of DNA strand cutting (19)(20)(21). We therefore predict that this mechanism might be able to deal with only a small number of DFSs, as our theory predicts (10). Previous studies have suggested that 53BP1 recognizes these aberrant structures in the cell cycle following underreplication.…”

“…1B). Theoretical analysis indicates that the distribution of origins in human cells is constrained to produce, on average, only a small number of DFSs and therefore indicates that human cells should possess a postreplicative mechanism capable of resolving these spontaneous events (10). Because a DFS will create a large segment of unreplicated DNA, our analyses suggest that humans and metazoans in general with significantly larger genomes than yeasts have evolved mechanisms to resolve them.…”

“…The average interorigin distance is ∼31 kb, consistent with initiation events being ∼100 kb apart (11,14,15) and ∼30% of origins being stochastically activated in any given S phase (6-9, 16, 17). Compared with yeast, human cells have an irregular distribution of origins with an unexpectedly high number of very large replicons (10). Using a mathematical approach that we have previously derived and validated (4) we estimate that one or two DFSs are expected to occur in every HeLa cell S phase (Fig.…”

“…This postreplicative mechanism may not be able to complete replication of all of the unreplicated DNA generated by DFSs, which may be hundreds of kilobases in size (10). It has been suggested that UFBs, which contain single-stranded DNA, might represent a mechanism for resolving partially replicated stretches of DNA (20,(26)(27)(28)(29) (dashed lines in Fig.…”