An Origin-Deficient Yeast Artificial Chromosome Triggers a Cell Cycle Checkpoint

Brabant, Anja J. van; Buchanan, Christina D.; Charboneau, Evonne J.; Fangman, Walton L.; Brewer, Bonita J.

doi:10.1016/s1097-2765(01)00216-7

Cited by 56 publications

(50 citation statements)

References 39 publications

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“…In this report, we demonstrate that sequences centromere-distal to replication gaps are lost at elevated frequency, suggesting that the gaps themselves are prone to breakage. This finding is consistent with the observation of Van Brabant et al (16,17) that a marker at the distal tip of the YAC used here was lost at a high frequency after ablation of human sequences serving as yeast origins. Also of interest is whether the consequences of incomplete replication vary across the genome, particularly with regard to repetitive versus nonrepetitive regions.…”

Section: Discussionsupporting

confidence: 93%

“…This yeast artificial chromosome (YAC) contains a 240-kb fragment of the human TCR-β (T-cell receptor, beta chain) region deleted for sequences that fortuitously behave as DNA replication origins in yeast (15)(16)(17). The replication of this YAC, which now depends on a strong yeast origin (ARS1) at the tip of the left arm, is markedly delayed, making the YAC genetically unstable and prone to large deletions due to incomplete replication (16,17). We analyzed G2-seq data from a strain containing this YAC using two complementary methods: (i) by comparing completion of replication along native chromosomes (e.g., chromosome XV, Fig.…”

Section: Resultsmentioning

confidence: 99%

“…For example, the region surrounding the strong early origin ARS1511 on chromosome XV completed replication before the end of S phase, the region to its left surrounding ARS1510 completed replication by early G2, and, by late G2, replication of the entire chromosome was complete. In contrast, [This host strain, which includes the cdc7-1 mutation, was chosen as a positive control to demonstrate the ability of our technique to identify late-replicating regions because it is identical to the previously published strain in which late replication of this YAC was originally reported (16).] (Left) Chromosome XV exhibits known pattern of origin firing in S phase, and its replication is complete by late G2; x axis shows positions (kb) on chromosome XV, and y axis shows relative read depth in the phase in question divided by read depth in G1, with both read depths normalized to genome-wide maxima.…”

Section: Resultsmentioning

confidence: 99%

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

Foss

Lao

Dalrymple

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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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...

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Section: Discussionsupporting

confidence: 93%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

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

Foss

Lao

Dalrymple

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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“…Indeed, 20 instances of the ARS consensus sequence can be found in our assembled genome, including 2 from the vector. This is an important consideration, because a 170-kb YAC lacking efficient origins of replication can invoke a cell-cycle checkpoint response in yeast (14).…”

Section: Discussionmentioning

confidence: 99%

One-step assembly in yeast of 25 overlapping DNA fragments to form a complete synthetic Mycoplasma genitalium genome

Gibson

Benders

Axelrod

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

411

318

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We previously reported assembly and cloning of the synthetic Mycoplasma genitalium JCVI-1.0 genome in the yeast Saccharomyces cerevisiae by recombination of six overlapping DNA fragments to produce a 592-kb circle. Here we extend this approach by demonstrating assembly of the synthetic genome from 25 overlapping fragments in a single step. The use of yeast recombination greatly simplifies the assembly of large DNA molecules from both synthetic and natural fragments.in vivo DNA assembly ͉ genome synthesis ͉ combinatorial assembly ͉ yeast transformation ͉ Mycoplasma genitalium ͉ synthetic biology Y east has long been considered a genetically tractable organism because of its ability to take up and recombine DNA fragments. More than 30 years ago, Hinnen et al. (1) reported the restoration of leucine biosynthesis in Saccharomyces cerevisiae by transformation of a leu2 -strain to LEU2ϩ using a method involving spheroplasts, CaCl 2 , and PEG. Soon after, Orr-Weaver et al. (2) reported mechanistic studies demonstrating that DNA molecules taken up during yeast transformation can integrate into yeast chromosomes through homologous recombination, and that the ends of the linear-transforming DNA are highly recombinogenic and react directly with homologous chromosomal sequences, whereas circular plasmids carrying yeast sequences integrate by a single crossover and only at low frequency. Subsequently, yeast transformation has become an indispensable tool in yeast genetics.Yeast recombination has since been applied to the construction of plasmids and yeast artificial chromosomes (YACs). In 1987, Ma et al. (3) constructed plasmids from two cotransformed DNA fragments containing homologous regions. In another process, called linker-mediated assembly, any DNA sequence can be joined to a vector DNA using short synthetic linkers that bridge the ends (4, 5). Similarly, four or five overlapping DNA pieces can be assembled and joined to vector DNA (4, 6, 7). This work demonstrated that yeast cells can take up multiple pieces of DNA, and that homologous yeast recombination is sufficiently efficient to correctly assemble the pieces into a single recombinant molecule.The limitations of assembly methods in yeast remain unknown. We recently assembled an entire synthetic M. genitalium genome using a combination of in vitro enzymatic recombination in early stages and in vivo yeast recombination in the final stage to produce the complete genome (8). In the first stage, overlapping Ϸ6-kb DNA cassettes were joined four at a time to form 25 Ϸ24-kb A-series assemblies. Three A-series assemblies were then joined to make 1/8 genome Ϸ72-kb B-series assemblies, and then two Bseries assemblies were assembled to make each of the Ϸ145-kb quarter-genome C-series assemblies. We accomplished the final assembly in yeast using three quarter-genome fragments and a fourth quarter fragment that had been cleaved by a restriction enzyme to provide a site for insertion of the vector DNA. Thus, some individual yeast cells have the capacity to simultaneously tak...

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“…34 In budding yeast, a system has been developed in which cells harbor an origin-deficient yeast artificial chromosome (YAC). 83 Replication of this YAC takes 50 minutes longer than an equivalent origin-proficient YAC due to the reduced number of efficiently firing origins. The extended period of ongoing DNA synthesis appears to prevent the onset of mitosis in these cells, yet further studies are needed to confirm that this delay is S-phase checkpoint-dependent.…”

Section: Budding Yeast Versus Higher Eukaryotesmentioning

confidence: 99%

DNA-Damage-Independent Checkpoints: Yeast and Higher Eukaryotes

Smith¹,

Giménez-Abián²,

Clarke³

2002

Cell Cycle

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An Origin-Deficient Yeast Artificial Chromosome Triggers a Cell Cycle Checkpoint

Cited by 56 publications

References 39 publications

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

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

One-step assembly in yeast of 25 overlapping DNA fragments to form a complete synthetic Mycoplasma genitalium genome

DNA-Damage-Independent Checkpoints: Yeast and Higher Eukaryotes

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