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

Abstract

Replication gaps that persist into mitosis likely represent important threats to genome stability, but experimental identification of these gaps has proved challenging. We have developed a technique that allows us to explore the dynamics by which genome replication is completed before mitosis. Using this approach, we demonstrate that excessive allocation of replication resources to origins within repetitive regions, induced by SIR2 deletion, leads to persistent replication gaps and genome instability. Conversely, the weakening of replication origins in repetitive regions suppresses these gaps. Given known age-and cancer-associated changes in chromatin accessibility at repetitive sequences, we suggest that replication gaps resulting from misallocation of replication resources underlie age-and disease-associated genome instability.SIR2 | DNA replication | repetitive sequences | replication gaps | ribosomal DNA S taggered initiation of DNA replication, which is common across eukaryotes from fungi to humans, means that, at any given time, in S phase, only a fraction of replication origins is activated. In recent years, a model has emerged to explain this pattern of DNA replication (1, 2). This model assumes that the pool of initiation factors required to fire licensed origins in S phase is limited, and therefore sufficient to fire only a subset of licensed origins at any given time. Licensed origins differ in their ability to recruit factors required for firing, so origins with higher affinity or accessibility fire earlier than those with lower affinity or accessibility. After activating the initial set of origins, firing factors are released, enabling the next set of origins to fire. This results in successive waves of origin activation. Genome replication eventually finishes when areas with the least accessible origins are replicated.In healthy human cells, the least accessible genomic regions consist of repetitive DNA, which represents about half of the genome and tends to be compacted into heterochromatin. However, recent studies suggest widespread opening of hetrochromatin during carcinogenesis and aging (3-5). Such reorganization of chromatin would expose a new suite of origins within repetitive DNA that could potentially compete initiation factors away from unique portions of the genome, thereby disrupting the normal genome-wide hierarchy of replication timing. Increased origin activity within repetitive DNA could thus compromise replication elsewhere in the genome. Given the limited pool of initiation factors, we propose that an increase in density of active origins within repetitive regions could result in a decreased density of such origins in unique regions of the genome, which, when combined with the stochastic nature of origin firing, may occasionally result in replication gaps, i.e., unique regions of the genome that are unable to complete replication before mitosis. This so-called "Random Replication Gap Problem" (RRGP) is the subject of long-standing speculation, but such gaps have never been experim...

Sir2 mitigates an intrinsic imbalance in origin licensing efficiency between early and late replicating euchromatin

“…Analyses of our S-phase Sort-Seq data revealed that Sir2 had a detectable effect on the replication progression of euchromatic origins, with later-euchromatic origins falling further behind both early-euchromatic origins and telomeric-X origins in sir2 Δ cells ( Figure 5 D ). These effects might be relevant to the inability to complete the duplication of late-replicating euchromatin by early-G2 phase in sir2 Δ cells as documented in (31). Therefore, the euchromatic- and telomeric-X origin cohorts were assessed using the data sets from this previous study.…”

“…Specifically, in sir2 Δ cells, the rDNA origins (r-ORIGIN) are more active, and because there are so many, limiting S-phase MCM complex activation factors get sequestered away from euchromatic origins causing a delay in euchromatic-origin activation. This sir2 Δ-caused sequestration of S-phase MCM complex activation factors contributes to the incomplete duplication of late replicating regions in G2 phase (31). In support of this model, sir2 Δ cells harboring a mutation within the rDNA origin, referred to here as r-origin* , reduces rDNA-dependent sequestration of S-phase MCM complex activation factors and partially rescues the delayed replication of late regions caused by sir2 Δ.…”

“…In sir2 Δ r-origin* cells, the duplication of the telomeric-X origin cohort in early G2 was substantially enhanced, such that the difference between early-euchromatic origins and telomeric-X-origins in early G2 no longer reached a significant P-value cut-off ( Figure 6A-B , compare early euchromatic and telomeric-X origins in early G2 in sir2 Δ r-ORIGIN cells to their relative behavior in sir2 Δ r-origin* cells). Therefore, the duplication of telomeric-X-origins in sir2 Δ cells was limited by the availability of S-phase MCM complex activation proteins sequestered by the rDNA array (31). However, in contrast, the late-euchromatic origin cohort remained under duplicated even in the sir2 Δ r-origin* cells ( Figure 6A-B , in particular compare sir2 Δ r-origin* to SIR2 r-ORIGIN ).…”

“…Direct assays of replication showed that the enhanced levels of MCM observed at early-euchromatic and telomeric-X origins in sir2 Δ cells correlated with enhanced replication activity of these elements, providing evidence that their origin activity was limited at the level of MCM complex loading. Finally, in the absence of Sir2, several regions of late-replicating euchromatin failed to complete duplication by the end of S-phase by a mechanism that was independent of the replication capacity of the Sir2-controlled rDNA locus that can indirectly alter euchromatic replication (30; 31). Thus, varying degrees of direct Sir2-mediated attenuation of MCM complex loading at individual origins balances the distribution of licensed origins across chromosomes to ensure the complete duplication of euchromatin by the end of S-phase.…”

The Miniature-chemostat Aging Device: A new experimental platform facilitates assessment of the transcriptional and chromatin landscapes of replicatively aging yeast

“…For example, instability at the ribosomal DNA (rDNA) locus, which contains ~100-200 tandemly repeated copies in the genome, can result in the formation of rDNA extra-chromosomal circles (ERCs), and is is a major determinant of yeast replicative lifespan (RLS) [11][12][13][14][15] . A single nucleotide polymorphism that reduces the rate of origin firing at the rDNA locus increases rDNA stability, and thereby increases RLS [16,17] . In a seemingly unrelated pathway, a decrease in vacuolar acidity early in a mother cell's life promotes mitochondrial dysfunction and limits her lifespan [7] .…”