Budding yeast Rap1, but not telomeric DNA, is inhibitory for multiple stages of DNA replication in vitro.
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ABSTRACT:
SUBMITTER: Douglas ME
PROVIDER: S-EPMC8191780 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The separation of DNA replication origin licensing and activation in the cell cycle is essential for genome stability across generations in eukaryotic cells. Pre-replicative complexes (pre-RCs) license origins by loading Mcm2-7 complexes in inactive form around DNA. During origin firing in S phase, replisomes assemble around the activated Mcm2-7 DNA helicase. Budding yeast pre-RCs have previously been reconstituted in vitro with purified proteins. Here, we show that reconstituted pre-RCs support replication of plasmid DNA in yeast cell extracts in a reaction that exhibits hallmarks of cellular replication initiation. Plasmid replication in vitro results in the generation of covalently closed circular daughter molecules, indicating that the system recapitulates the initiation, elongation, and termination stages of DNA replication. Unexpectedly, yeast origin DNA is not strictly required for DNA replication in vitro, as heterologous DNA sequences could support replication of plasmid molecules. Our findings support the notion that epigenetic mechanisms are important for determining replication origin sites in budding yeast, highlighting mechanistic principles of replication origin specification that are common among eukaryotes.
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Project description:G-quadruplexes are four-stranded nucleic acid structures involved in multiple cellular pathways including DNA replication and telomere maintenance. Such structures are formed by G-rich DNA sequences typified by telomeric DNA repeats. Whilst there is evidence for proteins that bind and regulate G-quadruplex formation, the molecular basis for this remains poorly understood. The budding yeast telomeric protein Rap1, originally identified as a transcriptional regulator functioning by recognizing double-stranded DNA binding sites, was one of the first proteins to be discovered to also bind and promote G-quadruplex formation in vitro. Here, we present the 2.4 Å resolution crystal structure of the Rap1 DNA-binding domain in complex with a G-quadruplex. Our structure not only provides a detailed insight into the structural basis for G-quadruplex recognition by a protein, but also gives a mechanistic understanding of how the same DNA-binding domain adapts to specifically recognize different DNA structures. The key observation is the DNA-recognition helix functions in a bimodal manner: In double-stranded DNA recognition one helix face makes electrostatic interactions with the major groove of DNA, whereas in G-quadruplex recognition a different helix face is used to make primarily hydrophobic interactions with the planar face of a G-tetrad.
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Project description:BackgroundDNA replication begins at specific locations called replication origins, where helicase and polymerase act in concert to unwind and process the single DNA filaments. The sites of active DNA synthesis are called replication forks. The density of initiation events is low when replication forks travel fast, and is high when forks travel slowly. Despite the potential involvement of epigenetic factors, transcriptional regulation and nucleotide availability, the causes of differences in replication times during DNA synthesis have not been established satisfactorily, yet.Methodology/principal findingsHere, we aimed at quantifying to which extent sequence properties contribute to the DNA replication time in budding yeast. We interpreted the movement of the replication machinery along the DNA template as a directed random walk, decomposing influences on DNA replication time into sequence-specific and sequence-independent components. We found that for a large part of the genome the elongation time can be well described by a global average replication rate, thus by a single parameter. However, we also showed that there are regions within the genomic landscape of budding yeast with highly specific replication rates, which cannot be explained by global properties of the replication machinery.Conclusion/significanceComputational models of DNA replication in budding yeast that can predict replication dynamics have rarely been developed yet. We show here that even beyond the level of initiation there are effects governing the replication time that can not be explained by the movement of the polymerase along the DNA template alone. This allows us to characterize genomic regions with significantly altered elongation characteristics, independent of initiation times or sequence composition.
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Project description:More than two decades of genetic research have identified and assigned main biological functions of shelterin proteins that safeguard telomeres. However, a molecular mechanism of how each protein subunit contributes to the protecting function of the whole shelterin complex remains elusive. Human Repressor activator protein 1 (Rap1) forms a multifunctional complex with Telomeric Repeat binding Factor 2 (TRF2). Rap1-TRF2 complex is a critical part of shelterin as it suppresses homology-directed repair in Ku 70/80 heterodimer absence. To understand how Rap1 affects key functions of TRF2, we investigated full-length Rap1 binding to TRF2 and Rap1-TRF2 complex interactions with double-stranded DNA by quantitative biochemical approaches. We observed that Rap1 reduces the overall DNA duplex binding affinity of TRF2 but increases the selectivity of TRF2 to telomeric DNA. Additionally, we observed that Rap1 induces a partial release of TRF2 from DNA duplex. The improved TRF2 selectivity to telomeric DNA is caused by less pronounced electrostatic attractions between TRF2 and DNA in Rap1 presence. Thus, Rap1 prompts more accurate and selective TRF2 recognition of telomeric DNA and TRF2 localization on single/double-strand DNA junctions. These quantitative functional studies contribute to the understanding of the selective recognition of telomeric DNA by the whole shelterin complex.
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Project description:We describe the DNA replication timing programs of 14 yeast mutants with an extended S phase identified by a novel genome-wide screen. These mutants are associated with the DNA replication machinery, cell-cycle control, and dNTP synthesis and affect different parts of S phase. In 13 of the mutants, origin activation time scales with the duration of S phase. A limited number of origins become inactive in these strains, with inactive origins characterized by small replicons and distributed throughout S phase. In sharp contrast, cells deleted of MRC1, a gene implicated in replication fork stabilization and in the replication checkpoint pathway, maintained wild-type firing times despite over twofold lengthening of S phase. Numerous dormant origins were activated in this mutant. Our data suggest that most perturbations that lengthen S phase affect the entire program of replication timing, rather than a specific subset of origins, maintaining the relative order of origin firing time and delaying firing with relative proportions. Mrc1 emerges as a regulator of this robustness of the replication program.
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Project description:Replication initiation and replication fork movement in the subtelomeric and telomeric DNA of native Y' telomeres of yeast were analyzed using two-dimensional gel electrophoresis techniques. Replication origins (ARSs) at internal Y' elements were found to fire in early-mid-S phase, while ARSs at the terminal Y' elements were confirmed to fire late. An unfired Y' ARS, an inserted foreign (bacterial) sequence, and, as previously reported, telomeric DNA each were shown to impose a replication fork pause, and pausing is relieved by the Rrm3p helicase. The pause at telomeric sequence TG(1-3) repeats was stronger at the terminal tract than at the internal TG(1-3) sequences located between tandem Y' elements. We show that the telomeric replication fork pause associated with the terminal TG(1-3) tracts begins approximately 100 bp upstream of the telomeric repeat tract sequence. Telomeric pause strength was dependent upon telomere length per se and did not require the presence of a variety of factors implicated in telomere metabolism and/or known to cause telomere shortening. The telomeric replication fork pause was specific to yeast telomeric sequence and was independent of the Sir and Rif proteins, major known components of yeast telomeric heterochromatin.
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Project description:Despite its evolutionarily conserved function in controlling DNA replication, the chromosomal binding sites of the budding yeast Rif1 protein are not well understood. Here, we analyse genome-wide binding of budding yeast Rif1 by chromatin immunoprecipitation, during G1 phase and in S phase with replication progressing normally or blocked by hydroxyurea. Rif1 associates strongly with telomeres through interaction with Rap1. By comparing genomic binding of wild-type Rif1 and truncated Rif1 lacking the Rap1-interaction domain, we identify hundreds of Rap1-dependent and Rap1-independent chromosome interaction sites. Rif1 binds to centromeres, highly transcribed genes and replication origins in a Rap1-independent manner, associating with both early and late-initiating origins. Interestingly, Rif1 also binds around activated origins when replication progression is blocked by hydroxyurea, suggesting association with blocked forks. Using nascent DNA labelling and DNA combing techniques, we find that in cells treated with hydroxyurea, yeast Rif1 stabilises recently synthesised DNA Our results indicate that, in addition to controlling DNA replication initiation, budding yeast Rif1 plays an ongoing role after initiation and controls events at blocked replication forks.
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Project description:Telomeric shelterin complex caps chromosome ends and plays a crucial role in telomere maintenance and protection. In the fission yeast Schizosaccharomyces pombe, shelterin is composed of telomeric single- and double-stranded DNA-binding protein subcomplexes Pot1-Tpz1 and Taz1-Rap1, which are bridged by their interacting protein Poz1. However, the structure of Poz1 and how Poz1 functions as an interaction hub in the shelterin complex remain unclear. Here we report the crystal structure of Poz1 in complex with Poz1-binding motifs of Tpz1 and Rap1. The crystal structure shows that Poz1 employs two different binding surfaces to interact with Tpz1 and Rap1. Unexpectedly, the structure also reveals that Poz1 adopts a dimeric conformation. Mutational analyses suggest that proper interactions between Tpz1, Poz1, and Rap1 in the shelterin core complex are required for telomere length homeostasis and heterochromatin structure maintenance at telomeres. Structural resemblance between Poz1 and the TRFH domains of other shelterin proteins in fission yeast and humans suggests a model for the evolution of shelterin proteins.
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Project description:Telomeres, the ends of linear eukaryotic chromosomes, have a specialized chromatin structure that provides a stable chromosomal terminus. In budding yeast Rap1 protein binds to telomeric TG repeat and negatively regulates telomere length. Here we show that binding of multiple Rap1 proteins stimulates DNA double-stranded break (DSB) induction at both telomeric and non-telomeric regions. Consistent with the role of DSB induction, Rap1 stimulates nearby recombination events in a dosage-dependent manner. Rap1 recruits Rif1 and Rif2 to telomeres, but neither Rif1 nor Rif2 is required for DSB induction. Rap1-mediated DSB induction involves replication fork progression but inactivation of checkpoint kinase Mec1 does not affect DSB induction. Rap1 tethering shortens artificially elongated telomeres in parallel with telomerase inhibition, and this telomere shortening does not require homologous recombination. These results suggest that Rap1 contributes to telomere homeostasis by promoting chromosome breakage.
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Project description:The chronological lifespan of eukaryotic organisms is extended by the mutational inactivation of conserved growth-signaling pathways that regulate progression into and through the cell cycle. Here we show that in the budding yeast S. cerevisiae, these and other lifespan-extending conditions, including caloric restriction and osmotic stress, increase the efficiency with which nutrient-depleted cells establish or maintain a cell cycle arrest in G1. Proteins required for efficient G1 arrest and longevity when nutrients are limiting include the DNA replication stress response proteins Mec1 and Rad53. Ectopic expression of CLN3 encoding a G1 cyclin downregulated during nutrient depletion increases the frequency with which nutrient depleted cells arrest growth in S phase instead of G1. Ectopic expression of CLN3 also shortens chronological lifespan in concert with age-dependent increases in genome instability and apoptosis. These findings indicate that replication stress is an important determinant of chronological lifespan in budding yeast. Protection from replication stress by growth-inhibitory effects of caloric restriction, osmotic and other stresses may contribute to hormesis effects on lifespan. Replication stress also likely impacts the longevity of higher eukaryotes, including humans.
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