A comprehensive genome-wide map of autonomously replicating sequences in a naive genome

Abstract

Eukaryotic chromosomes initiate DNA synthesis from multiple replication origins. The machinery that initiates DNA synthesis is highly conserved, but the sites where the replication initiation proteins bind have diverged significantly. Functional comparative genomics is an obvious approach to study the evolution of replication origins. However, to date, the Saccharomyces cerevisiae replication origin map is the only genome map available. Using an iterative approach that combines computational prediction and functional validation, we have generated a high-resolution genome-wide map of DNA replication origins in Kluyveromyces lactis. Unlike other yeasts or metazoans, K. lactis autonomously replicating sequences (KlARSs) contain a 50 bp consensus motif suggestive of a dimeric structure. This motif is necessary and largely sufficient for initiation and was used to dependably identify 145 of the up to 156 non-repetitive intergenic ARSs projected for the K. lactis genome. Though similar in genome sizes, K. lactis has half as many ARSs as its distant relative S. cerevisiae. Comparative genomic analysis shows that ARSs in K. lactis and S. cerevisiae preferentially localize to non-syntenic intergenic regions, linking ARSs with loci of accelerated evolutionary change.

Figure 1. Isolation and characterization of K. lactis ARSs.

(A) Schematic of the ARS screen in K. lactis. (B) Schematic for the iterative predict-and-verify approach for identifying genomic ARSs. (C) Logos of KlACS . Top panel, KlACS based on the initial 69 KlARSs. Bottom panel, KlACS based on 148 verified KlARSs. Arrows indicate an inverted repeat motif centered about an axis of symmetry marked by a vertical line. (D) An extended 50 bp motif of the S. cerevisiae ACS motif is shown in comparison with the KlACS.

Figure 2. A representation of genomic locations of KlARSs.

The 6 K. lactis chromosomes are shown in green with red blocks representing the KlARSs (KlARS size not to scale). The scale represents Megabases of genomic DNA.

Figure 3. Orientation of transcripts flanking S. cerevisiae and K. lactis ARSs.

ARSs from S. cerevisiae and K. lactis genomes are categorized with respect to flanking transcription. The headings display the transcription direction from the genes flanking the ARS.

Figure 4. KlACS is necessary and largely sufficient for KlARS function.

(A) Site-directed mutagenesis was used to replace trinucleotides in KlARS515. The mutant plasmids were tested for ARS activity. The mutated bases are underlined, and ARS function is indicated on the left and by the text color of the mutated nucleotides. The predicted KlACS motif is highlighted in blue. Mutants that did not affect function (+) are highlighted in black, while mutants affecting ARS function (+/− and −) are colored in red. Mutants that completely destroyed ARS function are indicated as (−), mutants that support the growth of minute colonies but contain dead cells on restreaking are indicated as (+/−) and mutants that had no effect are indicated as (+). Numbers on the right of the sequences are shown for reference. (B) Pictures of K. lactis transformed with mutant ARS plasmids on selective medium plates after 6 days of growth at 30°C. Plasmids with ARS activity comparable to the wild type plasmid (ARS+) are shown on the top row. Plasmids with weak ARS activity (ARS+/−) are shown in the middle row. Plasmids which show no visible ARS activity (ARS-) are on the bottom row. Numbers above the pictures correspond to mutant constructs from Figure 4A. Multiple clones of all plasmids were tested. Representative plasmids are shown. (C) ARS sequences of KlARS618, KlARS612 and KlARS524 were mutagenized to test the necessity of the predicted KlACS motif for ARS function. In all cases (6 total, see Table S1) the predicted mutation destroyed KlARS function. Each of the full KlARS sequences is 451bp. (D) DNA flanking the KlACS motif was truncated and the resulting sequences were tested for ARS function. The number of shortened KlARSs that retained function is shown on the right and Table S1.

Figure 5. Genomic replication initiation shown by 2D gel electrophoresis.

10 randomly chosen KlARSs and three non-ARS sequences were analyzed. The name of the ARS tested in its genomic context is indicated below each picture. Red arrows indicate “bubble arcs” caused by replication initiation events at probed loci. All experiments were performed on asynchronous log-phase cultures.

Figure 6. KARS12 and KARS101, two previously identified KlARSs.

(A) Overlapping ACSs of KARS12 (KlARS503) as predicted previously , (colored in green) and in this study (colored in blue). Substitution mutations are shown in lower case font and deletions in dashes. Mutations that disrupt ARS function are highlighted in red, and mutations that do not affect ARS function are highlighted in black. The logo of 50bp ACS is superimposed for reference. (+) indicates ARS function and (−) indicates no ARS function. (B) Complete overlapping ACSs (colored in blue) of KARS101 identified by linker substitution analysis and by motif search in this study. Octanucleotide linker mutations that destroyed ARS function (−) are indicated by red bars and those that compromised ARS function (+/−) are indicated by grey bars. The logo of 50bp ACS is superimposed for reference.