Novel features of ARS selection in budding yeast Lachancea kluyveri

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.

Figure 1

Screen to isolate L. kluyveri ARSs. (A) L. kluyveri genomic DNA was fragmented with MboI and ligated into the pIL07 vector. The resultant libraries were transformed into L. kluyveri strain FM628 and ARS plasmids were isolated from resulting colonies. (B) Representative colony sizes of plasmids showing ARS activity or the lack thereof.

Figure 2

L. kluyveri has a permissive mechanism of ARS selection relative to S. cerevisiae and K. lactis. ScARS [20], LkARS, and KlARS [20]plasmids were transformed into S. cerevisiae, L. kluyveri, and K. lactis and assayed for ARS function in the different species. The 'ARS source' column denotes the origin of the ARS, while the 'functions in' column denotes proportion of ARSs that are functional in the listed species. *: of the 80%, 20% of show weak ARS activity while 60% show strong ARS activity in L. kluyveri. 'WGD' denotes the whole genome duplication event leading to the S. cerevisiae lineage.

Figure 3

Identification of the LkACS motif. (A) Position Weighted Matrix logos of putative ACS motifs for S. cerevisiae, L. kluyveri and K. lactis. 'WGD' denotes the whole genome duplication event leading to the S. cerevisiae lineage. (B) Sequence logo of the statistically significant 30 bp motif found by GIMSAN in the set of 84 native L. kluyveri ARSs.

Figure 4

Truncation of LkARSs to narrow down essential functional regions. (A-C) Sub-fragments of three LkARSs (two more shown in Additional File 2, Figure S1) were cloned and tested for ARS function. Black boxes represent functional fragments. Red boxes represent non-functional fragments. For each ARS, the position of the best match to the 9 bp LkACS is indicated by a blue box. The extent of the truncation in basepairs is indicated on the left of the graphics (L = truncated from the left, R = truncated from the right). The length of the original full-length fragment isolated from the screen is indicated next to the first fragment from the top. *: This fragment retains very weak ARS activity.

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.

Figure 6

Extended sequence models. Graphical representation of the three linear weights models we studied that factor sequence information beyond the ACS. The paired linear model (A) is using an auxiliary motif in addition to the ACS PWM: the overall score is the weighted sum of the individual (disjoint) match scores. The contextual PWM model (B) consists of the weighted sum of the ACS match and the adjacent matches to the contextual PWMs. The latter are learned from the sites flanking the ACS sites in the alignment of the native ARSs. The Markov contextual model (C) combines the ACS match with the (log of the) Markov likelihood of the adjacent segments (normalized by an iid background model). The contextual Markov models are learned from the alignment of the native ARSs.