RESUMEN
Safeguards against excess DNA replication are often dysregulated in cancer, and driving cancer cells towards over-replication is a promising therapeutic strategy. We determined DNA synthesis patterns in cancer cells undergoing partial genome re-replication due to perturbed regulatory interactions (re-replicating cells). These cells exhibited slow replication, increased frequency of replication initiation events, and a skewed initiation pattern that preferentially reactivated early-replicating origins. Unlike in cells exposed to replication stress, which activated a novel group of hitherto unutilized (dormant) replication origins, the preferred re-replicating origins arose from the same pool of potential origins as those activated during normal growth. Mechanistically, the skewed initiation pattern reflected a disproportionate distribution of pre-replication complexes on distinct regions of licensed chromatin prior to replication. This distinct pattern suggests that circumventing the strong inhibitory interactions that normally prevent excess DNA synthesis can occur via at least two pathways, each activating a distinct set of replication origins.
Asunto(s)
Replicación del ADN , Origen de Réplica , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Ciclopentanos/farmacología , Replicación del ADN/efectos de los fármacos , Replicación del ADN/genética , Genoma Humano , Humanos , Mitosis/efectos de los fármacos , Modelos Biológicos , Pirimidinas/farmacología , Origen de Réplica/genéticaRESUMEN
Cell cycle progression in mammals is modulated by two ubiquitin ligase complexes, CRL4 and SCF, which facilitate degradation of chromatin substrates involved in the regulation of DNA replication. One member of the CRL4 complex, the WD-40 containing protein RepID (DCAF14/PHIP), selectively binds and activates a group of replication origins. Here we show that RepID recruits the CRL4 complex to chromatin prior to DNA synthesis, thus playing a crucial architectural role in the proper licensing of chromosomes for replication. In the absence of RepID, cells rely on the alternative ubiquitin ligase, SKP2-containing SCF, to progress through the cell cycle. RepID depletion markedly increases cellular sensitivity to SKP2 inhibitors, which triggered massive genome re-replication. Both RepID and SKP2 interact with distinct, non-overlapping groups of replication origins, suggesting that selective interactions of replication origins with specific CRL components execute the DNA replication program and maintain genomic stability by preventing re-initiation of DNA replication.
Asunto(s)
Cromatina/química , Proteínas Cullin/genética , Replicación del ADN , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas Quinasas Asociadas a Fase-S/genética , Ciclo Celular/efectos de los fármacos , Ciclo Celular/genética , Línea Celular Tumoral , Cromatina/metabolismo , Proteínas Cullin/metabolismo , Ciclopentanos/farmacología , Expresión Génica , Células HCT116 , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Células K562 , Osteoblastos/citología , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Unión Proteica , Transporte de Proteínas , Pirimidinas/farmacología , Origen de Réplica , Proteínas Quinasas Asociadas a Fase-S/antagonistas & inhibidores , Proteínas Quinasas Asociadas a Fase-S/metabolismo , Factor de Células Madre/genética , Factor de Células Madre/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Chromatin structure affects DNA replication patterns, but the role of specific chromatin modifiers in regulating the replication process is yet unclear. We report that phosphorylation of the human SIRT1 deacetylase on Threonine 530 (T530-pSIRT1) modulates DNA synthesis. T530-pSIRT1 associates with replication origins and inhibits replication from a group of 'dormant' potential replication origins, which initiate replication only when cells are subject to replication stress. Although both active and dormant origins bind T530-pSIRT1, active origins are distinguished from dormant origins by their unique association with an open chromatin mark, histone H3 methylated on lysine 4. SIRT1 phosphorylation also facilitates replication fork elongation. SIRT1 T530 phosphorylation is essential to prevent DNA breakage upon replication stress and cells harboring SIRT1 that cannot be phosphorylated exhibit a high prevalence of extrachromosomal elements, hallmarks of perturbed replication. These observations suggest that SIRT1 phosphorylation modulates the distribution of replication initiation events to insure genomic stability.
Asunto(s)
Replicación del ADN , Inestabilidad Genómica , Origen de Réplica , Sirtuina 1/metabolismo , Línea Celular , Roturas del ADN , Replicación del ADN/genética , Células HCT116 , Humanos , Células K562 , Células MCF-7 , Modelos Biológicos , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , ARN Interferente Pequeño/genética , Sirtuina 1/antagonistas & inhibidores , Sirtuina 1/genética , Treonina/química , Quinasas DyrKRESUMEN
DNA replication requires the recruitment of a pre-replication complex facilitated by Origin Recognition Complex (ORC) onto the chromatin during G1 phase of the cell cycle. The ORC-associated protein (ORCA/LRWD1) stabilizes ORC on chromatin. Here, we evaluated the genome-wide distribution of ORCA using ChIP-seq during specific time points of G1. ORCA binding sites on the G1 chromatin are dynamic and temporally regulated. ORCA association to specific genomic sites decreases as the cells progressed towards S-phase. The majority of the ORCA-bound sites represent replication origins that also associate with the repressive chromatin marks H3K9me3 and methylated-CpGs, consistent with ORCA-bound origins initiating DNA replication late in S-phase. Further, ORCA directly associates with the repressive marks and interacts with the enzymes that catalyze these marks. Regions that associate with both ORCA and H3K9me3, exhibit diminished H3K9 methylation in ORCA-depleted cells, suggesting a role for ORCA in recruiting the H3K9me3 mark at certain genomic loci. Similarly, DNA methylation is altered at ORCA-occupied sites in cells lacking ORCA. Furthermore, repressive chromatin marks influence ORCA's binding on chromatin. We propose that ORCA coordinates with the histone and DNA methylation machinery to establish a repressive chromatin environment at a subset of origins, which primes them for late replication.
Asunto(s)
Fase G1/genética , Heterocromatina/metabolismo , Proteínas de Microtúbulos/metabolismo , Origen de Réplica , Sitios de Unión , Línea Celular , Cromatina/metabolismo , Islas de CpG , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Metilación de ADN , Replicación del ADN , Código de Histonas , HumanosRESUMEN
In eukaryotes, genome duplication starts concomitantly at many replication initiation sites termed replication origins. The replication initiation program is spatially and temporally coordinated to ensure accurate, efficient DNA synthesis that duplicates the entire genome while maintaining other chromatin-dependent functions. Unlike in prokaryotes, not all potential replication origins in eukaryotes are needed for complete genome duplication during each cell cycle. Instead, eukaryotic cells vary the use of initiation sites so that only a fraction of potential replication origins initiate replication each cell cycle. Flexibility in origin choice allows each eukaryotic cell type to utilize different initiation sites, corresponding to unique nuclear DNA packaging patterns. These patterns coordinate replication with gene expression and chromatin condensation. Budding yeast replication origins share a consensus sequence that marks potential initiation sites. Metazoan origins, on the other hand, lack a consensus sequence. Rather, they are associated with a collection of structural features, chromatin packaging features, histone modifications, transcription, and DNA-DNA/DNA-protein interactions. These features confer cell type-specific replication and expression and play an essential role in maintaining genomic stability.
Asunto(s)
Replicación del ADN , Origen de Réplica/fisiología , Animales , Cromatina/genética , Cromatina/metabolismo , Eucariontes/genética , Células Eucariotas/fisiología , Inestabilidad Genómica/fisiología , Humanos , Saccharomycetales/genéticaRESUMEN
BACKGROUND: Eukaryotic genome duplication starts at discrete sequences (replication origins) that coordinate cell cycle progression, ensure genomic stability and modulate gene expression. Origins share some sequence features, but their activity also responds to changes in transcription and cellular differentiation status. RESULTS: To identify chromatin states and histone modifications that locally mark replication origins, we profiled origin distributions in eight human cell lines representing embryonic and differentiated cell types. Consistent with a role of chromatin structure in determining origin activity, we found that cancer and non-cancer cells of similar lineages exhibited highly similar replication origin distributions. Surprisingly, our study revealed that DNase hypersensitivity, which often correlates with early replication at large-scale chromatin domains, did not emerge as a strong local determinant of origin activity. Instead, we found that two distinct sets of chromatin modifications exhibited strong local associations with two discrete groups of replication origins. The first origin group consisted of about 40,000 regions that actively initiated replication in all cell types and preferentially colocalized with unmethylated CpGs and with the euchromatin markers, H3K4me3 and H3K9Ac. The second group included origins that were consistently active in cells of a single type or lineage and preferentially colocalized with the heterochromatin marker, H3K9me3. Shared origins replicated throughout the S-phase of the cell cycle, whereas cell-type-specific origins preferentially replicated during late S-phase. CONCLUSIONS: These observations are in line with the hypothesis that differentiation-associated changes in chromatin and gene expression affect the activation of specific replication origins.
RESUMEN
Chromosome replication, gene expression and chromatin assembly all occur on the same template, necessitating a tight spatial and temporal coordination to maintain genomic stability. The distribution of replication initiation events is responsive to local and global changes in chromatin structure and is affected by transcriptional activity. Concomitantly, replication origin sequences, which determine the locations of replication initiation events, can affect chromatin structure and modulate transcriptional efficiency. The flexibility observed in the replication initiation landscape might help achieve complete and accurate genome duplication while coordinating the DNA replication program with transcription and other nuclear processes in a cell-type specific manner. This review discusses the relationships among replication origin distribution, local and global chromatin structures and concomitant nuclear metabolic processes.