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Novel features of ARS selection in budding yeast Lachancea kluyveri.

Liachko I, Tanaka E, Cox K, Chung SC, Yang L, Seher A, Hallas L, Cha E, Kang G, Pace H, Barrow J, Inada M, Tye BK, Keich U - BMC Genomics (2011)

Bottom Line: Moreover, compared with S. cerevisiae and K. lactis, the replication licensing machinery in L. kluyveri seems more tolerant to variations in the ARS sequence composition.Our findings demonstrate a replication initiation system with novel features and underscore the functional diversity within the budding yeasts.Furthermore, we have developed new approaches for analyzing biologically functional DNA sequences with ill-defined motifs.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Mathematics and Statistics, University of Sydney, Sydney, Australia. bt16@cornell.edu

ABSTRACT

Background: The characterization of DNA replication origins in yeast has shed much light on the mechanisms of initiation of DNA replication. However, very little is known about the evolution of origins or the evolution of mechanisms through which origins are recognized by the initiation machinery. This lack of understanding is largely due to the vast evolutionary distances between model organisms in which origins have been examined.

Results: In this study we have isolated and characterized autonomously replicating sequences (ARSs) in Lachancea kluyveri - a pre-whole genome duplication (WGD) budding yeast. Through a combination of experimental work and rigorous computational analysis, we show that L. kluyveri ARSs require a sequence that is similar but much longer than the ARS Consensus Sequence well defined in Saccharomyces cerevisiae. Moreover, compared with S. cerevisiae and K. lactis, the replication licensing machinery in L. kluyveri seems more tolerant to variations in the ARS sequence composition. It is able to initiate replication from almost all S. cerevisiae ARSs tested and most Kluyveromyces lactis ARSs. In contrast, only about half of the L. kluyveri ARSs function in S. cerevisiae and less than 10% function in K. lactis.

Conclusions: Our findings demonstrate a replication initiation system with novel features and underscore the functional diversity within the budding yeasts. Furthermore, we have developed new approaches for analyzing biologically functional DNA sequences with ill-defined motifs.

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Mutagenesis of LkARSs to identify sequences necessary for LkARS function. (A-C) the shortest functional fragments of the three LkARSs in Figure 4 were mutated and tested for ARS function. The mutated residues are underlined. Mutations that disrupted ARS function are colored in red. The motif logos correspond to the best match of the predicted 9 bp LkACS and the relevant sequence is colored blue. (D) Representative examples of ARS function. LkARS-E139 mutant plasmids transformed into L. kluyveri and plated on selective media. The numbers correspond to mutant ARS fragments in (A). 'Empty' denotes the empty vector negative control, 'WT' denotes the full length LkARS-E139 positive control.
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Figure 5: Mutagenesis of LkARSs to identify sequences necessary for LkARS function. (A-C) the shortest functional fragments of the three LkARSs in Figure 4 were mutated and tested for ARS function. The mutated residues are underlined. Mutations that disrupted ARS function are colored in red. The motif logos correspond to the best match of the predicted 9 bp LkACS and the relevant sequence is colored blue. (D) Representative examples of ARS function. LkARS-E139 mutant plasmids transformed into L. kluyveri and plated on selective media. The numbers correspond to mutant ARS fragments in (A). 'Empty' denotes the empty vector negative control, 'WT' denotes the full length LkARS-E139 positive control.

Mentions: We isolated functional fragments of three of the LkARSs (LkARS-E139, LkARS-E848, and LkARS-C35) that are shorter than 100 bp and performed mutagenesis scanning experiments to identify specific sequences necessary for the ARS function of these DNA fragments (Figure 5). Using site-directed mutagenesis we systematically replaced tri-nucleotides every 8-11 bp within the functional LkARS fragments. The resulting mutants were tested for ARS function. In each of the three cases we identified a single mutant which destroyed ARS function (Figure 5).


Novel features of ARS selection in budding yeast Lachancea kluyveri.

Liachko I, Tanaka E, Cox K, Chung SC, Yang L, Seher A, Hallas L, Cha E, Kang G, Pace H, Barrow J, Inada M, Tye BK, Keich U - BMC Genomics (2011)

Mutagenesis of LkARSs to identify sequences necessary for LkARS function. (A-C) the shortest functional fragments of the three LkARSs in Figure 4 were mutated and tested for ARS function. The mutated residues are underlined. Mutations that disrupted ARS function are colored in red. The motif logos correspond to the best match of the predicted 9 bp LkACS and the relevant sequence is colored blue. (D) Representative examples of ARS function. LkARS-E139 mutant plasmids transformed into L. kluyveri and plated on selective media. The numbers correspond to mutant ARS fragments in (A). 'Empty' denotes the empty vector negative control, 'WT' denotes the full length LkARS-E139 positive control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3306766&req=5

Figure 5: Mutagenesis of LkARSs to identify sequences necessary for LkARS function. (A-C) the shortest functional fragments of the three LkARSs in Figure 4 were mutated and tested for ARS function. The mutated residues are underlined. Mutations that disrupted ARS function are colored in red. The motif logos correspond to the best match of the predicted 9 bp LkACS and the relevant sequence is colored blue. (D) Representative examples of ARS function. LkARS-E139 mutant plasmids transformed into L. kluyveri and plated on selective media. The numbers correspond to mutant ARS fragments in (A). 'Empty' denotes the empty vector negative control, 'WT' denotes the full length LkARS-E139 positive control.
Mentions: We isolated functional fragments of three of the LkARSs (LkARS-E139, LkARS-E848, and LkARS-C35) that are shorter than 100 bp and performed mutagenesis scanning experiments to identify specific sequences necessary for the ARS function of these DNA fragments (Figure 5). Using site-directed mutagenesis we systematically replaced tri-nucleotides every 8-11 bp within the functional LkARS fragments. The resulting mutants were tested for ARS function. In each of the three cases we identified a single mutant which destroyed ARS function (Figure 5).

Bottom Line: Moreover, compared with S. cerevisiae and K. lactis, the replication licensing machinery in L. kluyveri seems more tolerant to variations in the ARS sequence composition.Our findings demonstrate a replication initiation system with novel features and underscore the functional diversity within the budding yeasts.Furthermore, we have developed new approaches for analyzing biologically functional DNA sequences with ill-defined motifs.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Mathematics and Statistics, University of Sydney, Sydney, Australia. bt16@cornell.edu

ABSTRACT

Background: The characterization of DNA replication origins in yeast has shed much light on the mechanisms of initiation of DNA replication. However, very little is known about the evolution of origins or the evolution of mechanisms through which origins are recognized by the initiation machinery. This lack of understanding is largely due to the vast evolutionary distances between model organisms in which origins have been examined.

Results: In this study we have isolated and characterized autonomously replicating sequences (ARSs) in Lachancea kluyveri - a pre-whole genome duplication (WGD) budding yeast. Through a combination of experimental work and rigorous computational analysis, we show that L. kluyveri ARSs require a sequence that is similar but much longer than the ARS Consensus Sequence well defined in Saccharomyces cerevisiae. Moreover, compared with S. cerevisiae and K. lactis, the replication licensing machinery in L. kluyveri seems more tolerant to variations in the ARS sequence composition. It is able to initiate replication from almost all S. cerevisiae ARSs tested and most Kluyveromyces lactis ARSs. In contrast, only about half of the L. kluyveri ARSs function in S. cerevisiae and less than 10% function in K. lactis.

Conclusions: Our findings demonstrate a replication initiation system with novel features and underscore the functional diversity within the budding yeasts. Furthermore, we have developed new approaches for analyzing biologically functional DNA sequences with ill-defined motifs.

Show MeSH
Related in: MedlinePlus