Short DNA fragments without sequence similarity are initiation sites for replication in the chromosome of the yeast Yarrowia lipolytica

Abstract

We have previously shown that both a centromere (CEN) and a replication origin are necessary for plasmid maintenance in the yeast Yarrowia lipolytica (). Because of this requirement, only a small number of centromere-proximal replication origins have been isolated from Yarrowia. We used a CEN-based plasmid to obtain noncentromeric origins, and several new fragments, some unique and some repetitive sequences, were isolated. Some of them were analyzed by two-dimensional gel electrophoresis and correspond to actual sites of initiation (ORI) on the chromosome. We observed that a 125-bp fragment is sufficient for a functional ORI on plasmid, and that chromosomal origins moved to ectopic sites on the chromosome continue to act as initiation sites. These Yarrowia origins share an 8-bp motif, which is not essential for origin function on plasmids. The Yarrowia origins do not display any obvious common structural features, like bent DNA or DNA unwinding elements, generally present at or near eukaryotic replication origins. Y. lipolytica origins thus share features of those in the unicellular Saccharomyces cerevisiae and in multicellular eukaryotes: they are discrete and short genetic elements without sequence similarity.

Figure 1

Cloning of Y. lipolytica origins with a centromeric plasmid. (A) The cloning vector pINA732-Δ contains the LEU2 gene (dotted area), the CEN1 centromere, and flanking genomic sequences. Y. lipolytica genomic DNA digested with Sau3A was ligated into the BamHI site of the vector. (B) Characteristics of the eight ORI elements analyzed in this article. Demonstration that five of them are active origins comes from Vernis et al. (1997) and from data presented below in this article. We therefore adopt the ORI designation for all of them. Two of these sequences, marked by a star (ori1068 and ori3018), were subjected to systematic size reduction to determine the minimal functional fragment (Figure 4). The chromosomal localization of the different fragments was determined by pulsed-field gel electrophoresis and Southern blotting using reference strain E150. Chromosomal bands are numbered according to Vernis et al. (1997). Chromosomal activity (active; nt, not tested) refers to the observation of replication initiation signals after 2D gel electrophoresis.

Figure 2

Localization of the repeated oriX009 sequence in the genome of Y. lipolytica. (A) Map of a chromosomal integration at an oriX009 locus of the pINA989 containing oriX009 within its genomic environment, the LEU2 gene, and an I-SceI site. pBR322 vector sequences are shown in light gray. (B) Chromosomes from various transformants (lanes 1–5) were embedded in agarose plugs, digested with I-SceI, and separated under the following field inversion gel electrophoresis conditions: 1% pulsed field agarose (Bio-Rad, Bio-Rad, Hercules, CA) in 0.5× TAE buffer, run at 6 volts/cm for 14 h at 14°C with an angle of 106° and a variation of pulse from 0.41 sec to 2 min 6.52 sec. The DNA was transferred to a membrane and hybridized with a pBR322 probe (black box). A nontransformed strain is shown as a control in lane 6. The Raoul marker (Appligene, Heidelberg, Germany) is in lane 7. S. cerevisiae chromosomes and λ concatemers from Bio-Rad (not visible on the autoradiogram) were also used as molecular weight markers. As a comparison, the smallest chromosome among the six from Yarrowia is 2.6 Mb, and the largest is 4.9 Mb.

Figure 3

Ori4021 is active in the genome. (A) Sketch showing the migration patterns of various types of replication intermediates during neutral/neutral 2D gel electrophoresis. B) A restriction map of the genomic locus shows the two BamHI and BglII restriction fragments that are separated on a 2D gel and hybridized with an ori4021 probe. The pause signal observed along the Y arc on the BglII digest is not discussed here.

Figure 4

Mapping the minimal functional fragment of ori1068 (A) and ori3018 (B). Position and orientation of the synthetic primers are shown on the top line. Nucleotides positions are according to GenBank M91601 and M91600. The strain INAG3122 was transformed by electroporation with centromeric plasmids containing the reduced origins, because only replicative transformation is obtained at high frequency with this method. Results of transformation are shown by + and −. NR, Not recovered in E. coli.

Figure 5

Localization of the consensus sequence TDCAAGTH in six of the eight Y. lipolytica origins. Nucleotide sequences are deposited under the following GenBank accession numbers: AF038941 (oriX009), AF038942 (oriX096), AF038943 (ori4021), AF039581 (ori-rDNA), and AF038940 (ori3068).

Figure 6

Y. lipolytica origins do not contain structural motifs that are frequent in S. cerevisiae ARS elements. (A) S. cerevisiae ARS1 contains a strong, bent DNA region. The 2D projection of the 3D path of the molecule was calculated as described in MATERIALS AND METHODS. The position of the three elements A, B1, B2, and B3 (Marahrens and Stillman, 1992), the binding sites of ORC and ABF1 (Lee and Bell, 1997), and the site of initiation of DNA synthesis (Bielinsky and Gerbi, 1998) are indicated. (B) Y. lipolytica origins are not intrinsically curved. The 3D path of the minimal origin region of ori3018 (144 bp), ori1068 (125 bp), and oriX009 (159 bp) was calculated as described for ARS1. (C)Variation of helical stability and minor groove width along the S. cerevisiae ARS1 DNA fragment (350 bp). The free energy of the duplex or ΔG (207–227 kcal/mol; circles of increasing size) and the mean Roll angle (−1 to −0.1°; gray levels of increasing intensity) were calculated as described in MATERIALS AND METHODS for a 120-bp window sliding along the molecule. The position of the SAR mapped by Amati et al., 1988 (low Roll angle) and of the DUE (low ΔG) are indicated. (D) Spatial path and helical parameters of Y. lipolytica minimal ori-rDNA fragment (284 bp). The mean Roll angle varies from −0.3 to 0.5°, and the ΔG ranges from 215 to 226 kcal/mol. (E) Structural maps of large fragments containing S. cerevisiae HMRE ARS, Y. lipolytica ARS2, and Y. lipolytica ori4021. Like ARS1, the ACS of the HMRE ARS is flanked by a bent motif bearing binding sites for the auxiliary factors RAP1 (dark box) and ABF1 (open box) and by a DUE. The SAR fragment (Amati et al., 1988) maps perfectly to the region of low Roll angle. In all the graphs, the dotted lines correspond to the average value. Ori4021 is shown in its pBR322 environment, and the other two origins are at their chromosomal locus.

Figure 7

Ectopic initiation occurs within short DNA fragments. (A–C) Three different origins (A, ori-rDNA; B, oriX009; C, ori1068) were ligated into pINA214; the resulting plasmids were digested with BstXI and integrated into the LEU2 locus. The resulting genomic restriction map is shown, with the position of the sites used to digest the DNA for 2D gel analysis. The gels were blotted and hybridized with a pBR322 probe, indicated as black boxes. (D) The same experiment was performed with pINA214 as a control: hybridization of the 2D gel showed the absence of initiation at the LEU2 locus. Notice that the diagonal signal visible on these images is present on most chromosomal 2D gels performed in Yarrowia and may correspond to degradation products.