Various short autonomously replicating sequences from the yeast Kluyveromyces marxianus seemingly without canonical consensus

Abstract

Eukaryotic autonomously replicating sequences (ARSs) are composed of three domains, A, B, and C. Domain A is comprised of an ARS consensus sequence (ACS), while the B domain has the DNA unwinding element and the C domain is important for DNA-protein interactions. In Saccharomyces cerevisiae and Kluyveromyces lactis ARS101, the ACS is commonly composed of 11 bp, 5'-(A/T)AAA(C/T)ATAAA(A/T)-3'. This core sequence is essential for S. cerevisiae and K. lactis ARS activity. In this study, we identified ARS-containing sequences from genomic libraries of the yeast Kluyveromyces marxianus DMKU3-1042 and validated their replication activities. The identified K. marxianus DMKU3-1042 ARSs (KmARSs) have very effective replication ability but their sequences are divergent and share no common consensus. We have carried out point mutations, deletions, and base pairs substitutions within the sequences of some of the KmARSs to identify the sequence(s) that influence the replication activity. Consensus sequences same as the 11 bp ACS of S. cerevisiae and K. lactis were not found in all minimum functional KmARSs reported here except KmARS7. Moreover, partial sequences from different KmARSs are interchangeable among each other to retain the ARS activity. We have also specifically identified the essential nucleotides, which are indispensable for replication, within some of the KmARSs. Our deletions analysis revealed that only 21 bp in KmARS18 could retain the ARS activity. The identified KmARSs in this study are unique compared to other yeasts' ARSs, do not share common ACS, and are interchangeable.

Keywords: Functional validation; Interchanged sequences; KmARS; NHEJ; Transformability.

Fig. 1

The activity of short sequences of KmARSs. (A) Short sequences of different KmARSs ranging from 49 to 70 bp are shown (top panel). Seven KmARS sequences are fused to the ScURA3 gene at its 5ˊ end and the transformation efficacies of these constructs are depicted (bottom panel). Sequence alignment and logos of the short KmARSs are depicted in panel (B).

Fig. 2

Impact of truncations and nucleotides deletions on KmARS7. (A) KmARS7 (201-250) and its truncated fragments were fused at the 5ˊ end of the marker gene (top panel). A chart for the transformation efficacy of the regions KmARS7 (201-260) and KmARS7 (201-250) and its truncated fragments is depicted (bottom panel). (B) Sequences of KmARS7 (201-205) and its triple nucleotides deletion fragments are shown on the top and their corresponding transformation efficacies are depicted on the bottom. The values of transformation efficiencies (CFU μg⁻¹ DNA) in (A) and (B) are due to the use of different preparations of RAK3605 competent cells. Therefore, the charts in (A) and (B) represent the general patterns of transformability of KmARS7 (201-250) and its truncation and deletion products.

Fig. 3

Effect of 5′ and 3′ deletions on the activity of the region (46-105) of KmARS11. Nucleotide sequences of KmARS11 (46-105) and its deletion fragments are depicted on the top. A chart of transformation efficiencies of ARSs into the RAK3605 competent cells is shown on the bottom.

Fig. 4

Impact of nucleotides deletion on the activity of the regions KmARS18 (111-160) and KmARS18 (111-138). (A) Sequences of KmARS18 (111-160) and its successive deletion fragments are shown on the top. The downward arrow indicates the position that separates the two primers for each construct. The transformation efficacy of KmARS18 (111-160) and its deletion fragments are shown on the bottom. (B) Impact of single nucleotide deletion on the activity of KmARS18 (111-138). The sequences that produce highly efficient transformation (HET) are indicated by (+++), active regions after the deletion are indicated by (++), and the regions lost the activity are indicated by (-). The minimum sequence of KmARS18 with highly efficient transformability is indicated in yellow background. Other modifications presented are the replacement of GTC with CCA, the addition of A at position 131 of KmARS18 (111-138), deletion of G at position 122 of KmARS18 (111-138), and replacement of the nucleotides from 111-119 of KmARS18 (111-138) with sequences from KmARS7. These modifications are shown in sequences # 20, 21, 22, and 23, respectively.

Fig. 5

Effect of nucleotide substitutions and addition of cap sequences on the function of KmARS18 regions. (A) Influence of single nucleotide substitutions on the transformability of KmARS18 (111-138). Sequences with the symbol (+++) give a highly efficient transformation, those with the symbol (++) give moderate transformation, those with the symbol (+) give weak transformation, and those with the symbol (-) completely lost the activity. The sequence with (+/-) give variant transformability (mainly small colonies). (B) Sensitivity of KmARS18 (116-136) to cap. The addition of cap sequences at the end of KmARS18 (116-136) adversely affects the ARS function of this region. The addition of cap “cgcgc” to the region KmARS18 (111-159) positively enhanced the transformability, while the transformability of the capped KmARS18 (116-136) is greatly declined relative to the uncapped same region.

Fig. 6

Impact of truncations and nucleotides deletions on KmARS22 and KmARS36. (A) Effect of 5ˊ and 3ˊ ends truncations on KmARS22. Sequences of KmARS22 (991-1060) and its truncated fragments are shown on the top. These sequences are attached to the ScURA3 marker gene and the transformation efficiencies are shown on the bottom. (B) Influence of nucleotides deletions on KmARS36. The region KmARS36 (291-340) was divided into two primers as indicated by the downward arrow. Each primer was attached to one end of the ScURA3 marker gene. Sequences after successive deletions are shown (top panel). A chart for the transformation efficiency of the KmARS36 (291-340) and its deletion variants is shown (bottom panel).