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Investigating the role of the Est3 protein in yeast telomere replication.

ABSTRACT: The Est3 subunit of yeast telomerase, which adopts a predicted OB-fold, is essential for telomere replication. To assess the possible contributions that Est3 might make to enzyme catalysis, we compared telomerase activity from wild type and est3-Delta strains of Saccharomyces castellii, which revealed that loss of the Est3 subunit results in a 2- to 3-fold decline in nucleotide addition. This effect was not primer-specific, based on assessment of a panel of primers that spanned the template of the S. castellii telomerase RNA. Furthermore, using nuclear magnetic resonance chemical shift perturbation, no chemical shift change was observed at any site in the protein upon addition of single-stranded DNA, arguing against a role for Est3 in recognition of telomeric substrates by telomerase. Addition of exogenous Est3 protein, including mutant Est3 proteins that are severely impaired for telomere replication in vivo, fully restored activity in est3-Delta telomerase reactions. Thus, Est3 performs an in vivo regulatory function in telomere replication, which is distinct from any potential contribution that Est3 might make to telomerase activity.

SUBMITTER: Lee J

PROVIDER: S-EPMC2853109 | biostudies-literature | 2010 Apr

REPOSITORIES: biostudies-literature

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Investigating the role of the Est3 protein in yeast telomere replication.

Lee Jaesung J Mandell Edward K EK Rao Timsi T Wuttke Deborah S DS Lundblad Victoria V

Nucleic acids research 20100104 7

The Est3 subunit of yeast telomerase, which adopts a predicted OB-fold, is essential for telomere replication. To assess the possible contributions that Est3 might make to enzyme catalysis, we compared telomerase activity from wild type and est3-Delta strains of Saccharomyces castellii, which revealed that loss of the Est3 subunit results in a 2- to 3-fold decline in nucleotide addition. This effect was not primer-specific, based on assessment of a panel of primers that spanned the template of t ...[more]

PMID: 20047960

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Project description:BackgroundAlthough much is known about molecular mechanisms that prevent re-initiation of DNA replication on newly replicated DNA during a single cell cycle, knowledge is sparse regarding the regions that are most susceptible to re-replication when those mechanisms are bypassed and regarding the extents to which checkpoint pathways modulate re-replication. We used microarrays to learn more about these issues in wild-type and checkpoint-mutant cells of the fission yeast, Schizosaccharomyces pombe.ResultsWe found that over-expressing a non-phosphorylatable form of the replication-initiation protein, Cdc18 (known as Cdc6 in other eukaryotes), drove re-replication of DNA sequences genome-wide, rather than forcing high level amplification of just a few sequences. Moderate variations in extents of re-replication generated regions spanning hundreds of kilobases that were amplified (or not) approximately 2-fold more (or less) than average. However, these regions showed little correlation with replication origins used during S phase. The extents and locations of amplified regions in cells deleted for the checkpoint genes encoding Rad3 (ortholog of human ATR and budding yeast Mec1) and Cds1 (ortholog of human Chk2 and budding yeast Rad53) were similar to those in wild-type cells. Relatively minor but distinct effects, including increased re-replication of heterochromatic regions, were found specifically in cells lacking Rad3. These might be due to Cds1-independent roles for Rad3 in regulating re-replication and/or due to the fact that cells lacking Rad3 continued to divide during re-replication, unlike wild-type cells or cells lacking Cds1. In both wild-type and checkpoint-mutant cells, regions near telomeres were particularly susceptible to re-replication. Highly re-replicated telomere-proximal regions (50-100 kb) were, in each case, followed by some of the least re-replicated DNA in the genome.ConclusionThe origins used, and the extent of replication fork progression, during re-replication are largely independent of the replication and DNA-damage checkpoint pathways mediated by Cds1 and Rad3. The fission yeast pattern of telomere-proximal amplification adjacent to a region of under-replication has also been seen in the distantly-related budding yeast, which suggests that subtelomeric sequences may be a promising place to look for DNA re-replication in other organisms.

Dataset Information

Investigating the role of the Est3 protein in yeast telomere replication.

Publications

Investigating the role of the Est3 protein in yeast telomere replication.

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