DNA sequences and distinct mechanisms for ura4-595 and ura4-294 alleles of S. pombe

TL;DR

This paper identifies DNA sequence differences in two non-functional versions of the ura4 gene in fission yeast and suggests how these mutations occurred.

Contribution

The study provides new DNA sequence data and mutation mechanisms for two ura4 alleles in Schizosaccharomyces pombe.

Findings

The ura4-595 allele has a four bp duplication in the ORF due to DNA polymerase template slipping.

The ura4-294 allele has a nonsynonymous substitution likely caused by cytosine deamination.

Both alleles are functionally null and used for positive and negative selection in research.

Abstract

The
ura4
gene of the fission yeast
Schizosaccharomyces pombe
supports both positive and negative selection; consequently, this gene is widely employed as a powerful tool to study diverse biological processes. Here we report the DNA sequences of two functionally null alleles,
ura4-595
and
ura4-294
. The
ura4-595
allele has a four bp duplication of bp +63 to +66 (5’-CAAG-3’) within the ORF and the
ura4-294
allele has a nonsynonymous substitution (G to A) at bp +679. We infer that these alleles arose, respectively, by DNA polymerase template slipping and by nucleotide misincorporation (likely via cytosine deamination).

Figures

C](#f1) and 1D ). This type of mutation is most consistent with incorporation of the wrong nucleotide by the DNA polymerase, either directly or indirectly following spontaneous deamination of the corresponding cytosine base in the complementary DNA strand. Either way, this change leads to a single amino acid substitution in the encoded protein (Ura4-D227N) ( [Figure 1E](#f1) ). This change, which is localized to a highly conserved residue within a highly conserved region of the protein, is sufficient to inactivate the protein and render the cells auxotrophic for uracil ( [Figure 1B](#f1) ).

Tables

Oligonucleotides:
Name	Sequence
Ura4 FOR	5’- CCATCCCAGTTTAACTATGCTTCGTC-3’
Ura4 REV	5’- CGCCTAGGAAAACAAACGCAAACAA-3’

Fission yeast strains:
Name	Genotype	Source
WSP 0142	h - ura4-294	Smith strain GP31
WSP 0263	h + ura4-595 leu1-32	Smith strain GP191
WSP 0533	h - ura4-294	Gould strain KGY145
WSP 0556	h - ura4-D18	Gould strain KGY600
WSP 3776	h - ura4 +	This study
WSP 8537	h - ura4-595	This study

Full text

Description

The * ura4 * gene of fission yeast encodes a 264 amino acid long orotidine 5'-phosphate decarboxylase protein that is broadly and strongly conserved across taxa (Grimm

• et al.* 1988; Wood
• et al.* 2012). This enzyme catalyzes a key step in pyrimidine biosynthesis;

ura4 * mutants are unable to produce uracil de novo, but grow well if uracil is provided in the media. Reciprocally, if cells are provided with a substrate analog called 5-fluoroorotic acid (FOA), cells that express a functional Ura4 protein convert FOA to highly toxic 5-fluorourocil and are killed. Thus, the * ura4 * gene supports both positive selection (only * ura4 * wild-type cells will grow on media that lacks uracil) and negative selection (only * ura4 * mutants will grow on media that contains FOA). We sought to adapt this system to explore mechanisms of meiotic recombination and we reasoned that we could take advantage of previously defined alleles, ura4-595 and *ura4-294 * (Fox

• et al.* 1997). We therefore obtained strains harboring those alleles from the authors and, for the sake of independent confirmation, from an additional laboratory. As expected, haploid cells with the ura4-595 and ura4-294 alleles were auxotrophic for uracil and resistant to FOA ( ** Figure 1B ** ). However, the DNA sequences that we obtained—and which we confirmed by sequencing both strands of each allele and by sequencing alleles from different laboratories—differed from those reported previously.

The ura4-595 allele purportedly had a duplication of GATC at bp position 595 (Fox

• et al.* 1997). However, there is no GATC in that position within wild-type

ura4 * . Moreover, our analyses revealed that the ura4-595 allele actually harbors a four bp duplication of bp +63 to +66 (5’-CAAG-3’) within the ORF (these coordinates are numbered relative to the first nucleotide of the start codon) ( ** Figure 1C ** and 1D ). This type of mutation is most consistent with a template slipping mechanism during DNA replication, whereby the DNA polymerase loses its register on the template strand, backs up a short way, then resumes elongation from the new register. The four bp duplication leads to a frameshift for translation, resulting in a truncated protein whose sequence is scrambled for 32 amino acids beyond the lysine at residue 22 ( ** Figure 1E ** ). Correspondingly, the mutant cells lack a functional Ura4 protein and are auxotrophic for uracil ( ** Figure 1B ** ).

The ura4-294 allele purportedly had a C to T mutation at position 1212 (Fox

• et al.* 1997). However, our analyses revealed that the ura4-294 allele actually contains a G to A mutation at position +679 ( ** Figure 1C ** and 1D ). This type of mutation is most consistent with incorporation of the wrong nucleotide by the DNA polymerase, either directly or indirectly following spontaneous deamination of the corresponding cytosine base in the complementary DNA strand. Either way, this change leads to a single amino acid substitution in the encoded protein (Ura4-D227N) ( ** Figure 1E ** ). This change, which is localized to a highly conserved residue within a highly conserved region of the protein, is sufficient to inactivate the protein and render the cells auxotrophic for uracil ( ** Figure 1B ** ).

Methods

Strains of the indicated genotypes were constructed and propagated using standard fission yeast methods. Genomic DNA samples were prepared using smash and grab method with cells from 5 ml of culture. PCR and DNA sequencing were conducted using the listed oligonucleotide primers.

Reagents

**: **

**: **