Role for topoisomerase 1 in transcription-associated mutagenesis in yeast
Malcolm J. LippertNayun KimJang-Eun ChoRyan LarsonNathan E. SchoenlyShannon O’SheaSue Jinks-Robertson
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Abstract:
High levels of transcription in Saccharomyces cerevisiae are associated with increased genetic instability, which has been linked to DNA damage. Here, we describe a pGAL-CAN1 forward mutation assay for studying transcription-associated mutagenesis (TAM) in yeast. In a wild-type background with no alterations in DNA repair capacity, ≈50% of forward mutations that arise in the CAN1 gene under high-transcription conditions are deletions of 2–5 bp. Furthermore, the deletions characteristic of TAM localize to discrete hotspots that coincide with 2–4 copies of a tandem repeat. Although the signature deletions of TAM are not affected by the loss of error-free or error-prone lesion bypass pathways, they are completely eliminated by deletion of the TOP1 gene, which encodes the yeast type IB topoisomerase. Hotspots can be transposed into the context of a frameshift reversion assay, which is sensitive enough to detect Top1-dependent deletions even in the absence of high transcription. We suggest that the accumulation of Top1 cleavage complexes is related to the level of transcription and that their removal leads to the signature deletions. Given the high degree of conservation between DNA metabolic processes, the links established here among transcription, Top1, and mutagenesis are likely to extend beyond the yeast system.Keywords:
Transcription
ABSTRACT Mutations in the SPT3 gene were isolated as one class of suppressors of Ty and solo δ insertion mutations in Saccharomyces cerevisiae. Previous work has shown that null mutations in SPT3 abolish the normal Ty δ-δ transcript; instead, a transcript that initiates 800 bases farther downstream is made, suggesting that SPT3 is required for transcription initiation in δ sequences. We have selected for new spt mutations and have screened for those with the unique suppression pattern of spt3 mutations with respect to two insertion mutations. Our selection and screen has identified two additional genes, SPT7 and SPT8, that are also required for transcription initiation in δ sequences. We show that mutations in SPT7 or SPT8 result in the same alteration of Ty transcription as do mutations in SPT3. In addition, mutations in all three genes cause a sporulation defect. By assay of a Ty-lacZ fusion we have shown that spt3, spt7 and spt8 mutations reduce transcription from a δ sequence by 10-25-fold. Finally, we show that SPT3 mRNA levels are unaffected in either spt7 or spt8 mutants, suggesting that these two genes do not regulate transcription of SPT3.
Transcription
Trans-acting
Structural gene
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Lac repressor
Sequence (biology)
Indel
INDEL Mutation
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Transcription
Human genetics
Ribosomal protein
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ABSTRACT Mating type interconversion in Saccharomyces cerevisiae occurs by transposition of copies of the a or α mating type cassettes from inactive loci, HML and HMR, to an active locus, MAT. The lack of expression of the a and α genes at the silent loci results from repression by trans-acting regulators encoded by SIR (Silent Information Regulator) genes. In this paper we present evidence for the existence of four SIRgenes. Inactivation of any of these genes leads to expression of cassettes at both HML and HMR. Unusual complementation properties are observed for a number of sir mutations. Specifically, some recessive mutations in different genes fail to complement. The correspondence between SIR1, SIR2, SIR3, SIR4 and other genes with similar roles (MAR, CMT, STE8 and STE9) is presented.
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Epidermolytic palmoplantar keratoderma (EPPK) is an autosomal dominant genodermatosis caused by variants of keratin 9 (KRT9) or KRT1 gene. In this study causative gene mapping in a Chinese EPPK family was performed with Two-point linkage analysis and haplotyping. Positive linkage results were obtained on 17q (Zmax=2.06, θmax=0.0) at D17S799, which indicated KRT9 to be the most responsible gene for the family. Subsequently, direct sequencing identified a novel frameshift mutation caused by a 5bp deletion (∆GGAGG) in KRT9 in all affected individuals but not in the unaffected members or the 50 unrelated controls. The frameshift changed the encoding of the following nine amino acids and resulted in a readthrough translation in exon 7. The data revealed that the novel frameshift mutation in KRT9 was responsible for the Chinese EPPK pedigree. The researchers’ findings broaden the spectrum of KRT9 variants and provide further evidence for the highly genetic heterogeneity of EPPK.
Genodermatosis
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While characterizing the hsp70 gene family from Caenorhabditis elegans we encountered an unusual member of this family. Sequence data reveal that the hsp-2ps gene is a pseudogene of the constitutively expressed, heat-inducible hsp-1 gene. Two stop codons generated near the 5′ end of the sequence as well as several frameshift mutations and a large internal deletion confirm the identification of hsp-2ps as a pseudogene. The nucleotide substitution rate of the third codon position was twice that of the first and second codon positions, suggesting that the hsp-2ps gene was nonfunctional since the time of the duplication event. The hsp-2ps gene duplicates a region of the hsp-1 gene that lies exclusively within the transcribed region and retains the introns. We feel that the hsp-2ps gene was produced by a transpositional duplication event, which occurred approximately 8.5 million years ago.Key words: heat shock, pseudogene, Caenorhabditis elegans, hsp70.
Pseudogene
Stop codon
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ABSTRACT ICR-170-induced mutations in the CYC1 gene of the yeast Saccharomyces cerevisiae were investigated by genetic and DNA sequence analyses. Genetic analysis of 33 cyc1 mutations induced by ICR-170 and sequence analysis of eight representatives demonstrated that over one-third were frameshift mutations that occurred at one site corresponding to amino acid positions 29-30, whereas the remaining mutations were distributed more-or-less randomly, and a few of these were not frameshift mutations. The sequence results indicate that ICR-170 primarily induces G·C additions at sites containing monotonous runs of three G·C base pairs. However, some (see PDF) sites within the CYC1 gene were not mutated by ICR-170. Thus, ICR-170 is a relatively specific mutagen that preferentially acts on certain sites with monotonous runs of G·C base pairs.
Sequence (biology)
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Arabidopsis DNA hypomethylation mutation, ddm1 , results in a variety of developmental abnormalities by slowly inducing heritable lesions at unlinked loci. Here, late‐flowering traits observed at high frequencies in independently‐established ddm1 lines were genetically characterized. In all of the four late‐flowering lines examined the traits were dominant and mapped to the same chromosomal region, which is close or possibly identical to the FWA locus. The ddm1 ‐induced phenotypic onsets are apparently not random mutation events, but specific to a group of genes, suggesting the underlying epigenetic mechanism. The DNA methylation mutant provide useful system for identifying epigenetically‐regulated genes important for plant development.
RNA-Directed DNA Methylation
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Deficiency of the cytochrome P-450 steroid 21-hydroxylase (21-OHase)
which causes Congenital Adrenal Hyperplasia (CAH) is a monogenic autosomal
recessive disorder which is linked to HLA. There are two 21-OHase
genes in man, A and B, and they are mapped to the HLA class III region ~
3 kb 3' to the complement genes C4A and C4B, respectively. Two genes
encoding 21-OHase were isolated, characterized and sequenced. Both
21-OHase genes are ~ 3.3 kb in length and are split into 10 exons by
nine introns. Comparison of the two genes showed that although they are
highly conserved, there are three deleterious mutations in the 21-OHase
A gene which cause frameshifts and introduce in phase premature termination
codons. Thus the 21-OHase A gene is a pseudogene.
Comparison of the 21-OHase B gene to the other cytochrome P-450 sequences
revealed that although the cysteine-429 was conserved in 21-OHase,
there is very little homology with other cytochrome P-450, indicating it
belongs to a separate family of genes within the superfamily. Clear
evidence of polymorphism in 21-OHase is apparent on comparison with
other 21-OHase B sequences. There is a size polymorphism of 494 and 495
amino acids.
The differing severities of 21-OHase deficiency in CAH may be due to
allelic variants of the 21-OHase B gene, since in most cases the defect
is not due to gene deletion (Rumsby et al., 1986). A 21-OHase B gene
from a patient with CAH was characterized and sequenced. There were 13
nucleotide alterations in his single 21-OHase B gene, one of which at
codon 269 caused a serine to change to a threonine residue.
The G → C transversion in the 21-OHase B gene from the patient at codon
269 introduced a new NcoI restriction site into the gene. This restriction
fragment length polymorphism (RFLP) was used to study other patients
with CAH and normal individuals. The NcoI RFLP was found not to
be confined to the 21-OHase B gene but was also present in some 21-OHase
A genes. It is likely therefore that the mutation occurred in the
pseudogene first and then transferred to some 21-OHase B genes.
Pseudogene
C4A
Homology
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