Suppressible and nonsuppressible +1 G-C base pair insertions induced by ICR-170 at the his4 locus in Saccharomyces cerevisiae.

Fifteen independent ICR-170-induced his4 mutations in Saccharomyces cerevisiae were examined by DNA sequence analysis. All of the mutations contained a +1 G-C base pair addition in the HIS4 coding region. Eleven different sites of insertion were identified. Combined with previous DNA sequence data, 21 ICR-170-induced his4 mutations distributed at 16 different sites were analyzed. The insertions were always located in a consecutive run of two or more G-C base pairs, with all base pairs in each run having identical orientation. Long consecutive G-C runs were preferred target sites over short runs. Although some consecutive G-C runs appeared to be preferred target sites over others of identical length, such preference was not due to any particular type of nucleotide pair immediately adjacent to a given target site. In addition, DNA sequence analyses of the his4 mutations provided a basis for examining the mechanism of mRNA sequence recognition by extragenic suppressors of ICR-170-induced mutations. The implications of these results for mechanisms of frameshift suppression are discussed.

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Highly mutable sites for ICR-170-induced frameshift mutations are associated with potential DNA hairpin structures: studies with SUP4 and other Saccharomyces cerevisiae genes

Molecular and Cellular Biology ◽

10.1128/mcb.6.12.4425-4432.1986 ◽

1986 ◽

Vol 6 (12) ◽

pp. 4425-4432

Author(s):

D M Hampsey ◽

R A Koski ◽

F Sherman

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Sequences ◽

Base Pair ◽

Temporal Stability ◽

Loop Formation ◽

Induced Mutations ◽

Base Pairs ◽

Yeast Saccharomyces Cerevisiae ◽

Mutagen Specificity ◽

Hairpin Structures

The majority of the mutations induced by ICR-170 in both the CYC1 gene (J. F. Ernst et al. Genetics 111:233-241, 1985) and the HIS4 gene (L. Mathison and M. R. Culbertson, Mol. Cell. Biol. 5:2247-2256, 1985) of the yeast Saccharomyces cerevisiae were recently shown to be single G . C base-pair insertions at monotonous runs of two or more G . C base pairs. However, not all sites were equally mutable; in both the CYC1 and HIS4 genes there is a single highly mutable site where a G . C base pair is preferentially inserted at a [sequence in text]. Here we report the ICR-170 mutagen specificity at the SUP4-o tyrosine tRNA gene of yeast. Genetic fine structure analysis and representative DNA sequence determination of ICR-170-induced mutations revealed that there is also a single highly mutable site in SUP4-o and that the mutation is a G . C base-pair insertion at a monotonous run of G . C base pairs. Analysis of DNA sequences encompassing the regions of highly mutable sites for all three genes indicated that the mutable sites are at the bases of potential hairpin structures; this type of structure could not be found at any of the other, less mutable G . C runs in SUP4, CYC1, and HIS4. Based on these results and recent information regarding novel DNA structural conformations, we present a mechanism for ICR-170-induced mutagenesis. (i) ICR-170 preferentially binds to DNA in the beta conformation; factors that increase the temporal stability of this structure, such as adjacent stem-and-loop formation, increase the frequency of ICR-170 binding; (ii) the observed mutagen specificity reflects formation of a preferred ICR-170 intercalative geometry at [sequence in text] sites; (iii) during replication or repair, ICR-170 remains associated with the single-stranded template; (iv) stuttering or strand slippage by the polymerization complex as it encounters the mutagen results in nucleotide duplication; (v) subsequent replication or mismatch repair fixes the insertion into the genome. This mechanism accounts for both the IRC-170 mutagenic specificity and the molecular basis of the highly mutable sites in S. cerevisiae.

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Highly mutable sites for ICR-170-induced frameshift mutations are associated with potential DNA hairpin structures: studies with SUP4 and other Saccharomyces cerevisiae genes.

Molecular and Cellular Biology ◽

10.1128/mcb.6.12.4425 ◽

1986 ◽

Vol 6 (12) ◽

pp. 4425-4432 ◽

Cited By ~ 10

Author(s):

D M Hampsey ◽

R A Koski ◽

F Sherman

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Sequences ◽

Base Pair ◽

Temporal Stability ◽

Loop Formation ◽

Induced Mutations ◽

Base Pairs ◽

Yeast Saccharomyces Cerevisiae ◽

Mutagen Specificity ◽

Hairpin Structures

The majority of the mutations induced by ICR-170 in both the CYC1 gene (J. F. Ernst et al. Genetics 111:233-241, 1985) and the HIS4 gene (L. Mathison and M. R. Culbertson, Mol. Cell. Biol. 5:2247-2256, 1985) of the yeast Saccharomyces cerevisiae were recently shown to be single G . C base-pair insertions at monotonous runs of two or more G . C base pairs. However, not all sites were equally mutable; in both the CYC1 and HIS4 genes there is a single highly mutable site where a G . C base pair is preferentially inserted at a [sequence in text]. Here we report the ICR-170 mutagen specificity at the SUP4-o tyrosine tRNA gene of yeast. Genetic fine structure analysis and representative DNA sequence determination of ICR-170-induced mutations revealed that there is also a single highly mutable site in SUP4-o and that the mutation is a G . C base-pair insertion at a monotonous run of G . C base pairs. Analysis of DNA sequences encompassing the regions of highly mutable sites for all three genes indicated that the mutable sites are at the bases of potential hairpin structures; this type of structure could not be found at any of the other, less mutable G . C runs in SUP4, CYC1, and HIS4. Based on these results and recent information regarding novel DNA structural conformations, we present a mechanism for ICR-170-induced mutagenesis. (i) ICR-170 preferentially binds to DNA in the beta conformation; factors that increase the temporal stability of this structure, such as adjacent stem-and-loop formation, increase the frequency of ICR-170 binding; (ii) the observed mutagen specificity reflects formation of a preferred ICR-170 intercalative geometry at [sequence in text] sites; (iii) during replication or repair, ICR-170 remains associated with the single-stranded template; (iv) stuttering or strand slippage by the polymerization complex as it encounters the mutagen results in nucleotide duplication; (v) subsequent replication or mismatch repair fixes the insertion into the genome. This mechanism accounts for both the IRC-170 mutagenic specificity and the molecular basis of the highly mutable sites in S. cerevisiae.

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DNA sequence analysis of spontaneous mutations in the SUP4-o gene of Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.8.2.978-981.1988 ◽

1988 ◽

Vol 8 (2) ◽

pp. 978-981

Author(s):

C N Giroux ◽

J R Mis ◽

M K Pierce ◽

S E Kohalmi ◽

B A Kunz

Keyword(s):

Saccharomyces Cerevisiae ◽

Sequence Analysis ◽

Transposable Elements ◽

Dna Sequencing ◽

Dna Sequence ◽

Base Pair ◽

Dna Sequence Analysis ◽

Spontaneous Mutations ◽

Yeast Saccharomyces Cerevisiae ◽

Spontaneous Mutagenesis

A collection of 196 spontaneous mutations in the SUP4-o gene of the yeast Saccharomyces cerevisiae was analyzed by DNA sequencing. The classes of mutation identified included all possible types of base-pair substitution, deletions of various lengths, complex alterations involving multiple changes, and insertions of transposable elements. Our findings demonstrate that at least several different mechanisms are responsible for spontaneous mutagenesis in S. cerevisiae.

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RAD3 gene of Saccharomyces cerevisiae: nucleotide sequence of wild-type and mutant alleles, transcript mapping, and aspects of gene regulation

Molecular and Cellular Biology ◽

10.1128/mcb.5.1.17-26.1985 ◽

1985 ◽

Vol 5 (1) ◽

pp. 17-26

Author(s):

L Naumovski ◽

G Chu ◽

P Berg ◽

E C Friedberg

Keyword(s):

Saccharomyces Cerevisiae ◽

Nucleotide Sequence ◽

Coding Region ◽

Base Pairs ◽

S1 Nuclease ◽

Calculated Molecular Weight ◽

Base Pair Deletion ◽

S1 Nuclease Mapping ◽

Essential Function ◽

Nuclease Mapping

We determined the complete nucleotide sequence of the RAD3 gene of Saccharomyces cerevisiae. The coding region of the gene contained 2,334 base pairs that could encode a protein with a calculated molecular weight of 89,796. Analysis of RAD3 mRNA by Northern blots and by S1 nuclease mapping indicated that the transcript was approximately 2.5 kilobases and did not contain intervening sequences. Fusions between the RAD3 gene and the lac'Z gene of Escherichia coli were constructed and used to demonstrate that the RAD3 gene was not inducible by DNA damage caused by UV radiation or 4-nitroquinoline-1-oxide. Two UV-sensitive chromosomal mutant alleles of RAD3, rad3-1 and rad3-2, were rescued by gap repair of a centromeric plasmid, and their sequences were determined. The rad3-1 mutation changed a glutamic acid to lysine, and the rad3-2 mutation changed a glycine to arginine. Previous studies have shown that disruption of the RAD3 gene results in loss of an essential function and is associated with inviability of haploid cells. In the present experiments, plasmids carrying the rad3-1 and rad3-2 mutations were introduced into haploid cells containing a disrupted RAD3 gene. These plasmids expressed the essential function of RAD3 but not its DNA repair function. A 74-base-pair deletion at the 3' end of the RAD3 coding region or a fusion of this deletion to the E. coli lac'Z gene did not affect either function of RAD3.

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Repetitive Dictyostelium heat-shock promotor functions in Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.4.4.591-598.1984 ◽

1984 ◽

Vol 4 (4) ◽

pp. 591-598

Author(s):

J Cappello ◽

C Zuker ◽

H F Lodish

Keyword(s):

Saccharomyces Cerevisiae ◽

Heat Shock ◽

Nucleotide Sequence ◽

Dna Sequence ◽

Base Pair ◽

Shock Treatment ◽

Yeast Cells ◽

Genomic Segment ◽

Heat Shock Treatment ◽

Shock Response

The Dictyostelium genome contains 40 copies of a 4.7-kilobase repetitive and apparently transposable DNA sequence (DIRS-1) and about 250 smaller elements that appear to be deletions or rearrangements of DIRS-1. Transcripts of these sequences are induced during differentiation and also by heat shock treatment of growing cells. We showed that one such cloned element, pB41.6 (2.5 kilobases) contains a nucleotide sequence identical to the Drosophila consensus heat shock promotor. To test whether this sequence might indeed control the expression of DIRS-1-related RNAs, we have cloned this genomic segment into yeast cells. In yeast cells, 41.6 directs synthesis of a 1.7-kilobase RNA that is induced at least 10-fold by heat shock. Transcription initiates at about 124 bases 3' of the putative promotor sequence and terminates within the 41.6 insert. A 381-base-pair subclone that contains the putative promotor sequence is sufficient to induce the heat shock response of 41.6 in yeast cells.

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Repair of heteroduplex plasmid DNA after transformation into Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.6.10.3401-3409.1986 ◽

1986 ◽

Vol 6 (10) ◽

pp. 3401-3409

Author(s):

D K Bishop ◽

R D Kolodner

Keyword(s):

Saccharomyces Cerevisiae ◽

Plasmid Dna ◽

Base Pair ◽

Base Pairs ◽

Plasmid Dnas ◽

Repair Efficiency ◽

Single Insertion ◽

Base Pair Insertion

Purified heteroduplex plasmid DNAs containing 8- or 12-base-pair insertion mismatches or AC or CT substitution mismatches were used to transform Saccharomyces cerevisiae. Two insertion mismatches, separated by 943 base pairs, were repaired independently of each other at least 55% of the time. This suggested that repair tracts were frequently shorter than 1 kilobase. The two insertion mismatches were repaired with different efficiencies. Comparison of the repair efficiency of one mismatched site with or without an adjacent mismatch suggests that mismatches promote their own repair and can influence the repair of neighboring mismatches. When two different plasmids containing single-insertion mismatches were transformed into S. cerevisiae cells, a slight preference towards insertion was detected among repair products of one of the two plasmids, while no repair preference was detected among transformants with the second plasmid.

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Supercoiling DNA locates mismatches

10.1101/130567 ◽

2017 ◽

Author(s):

Andrew Dittmore ◽

Sumitabha Brahmachari ◽

Yasuhara Takagi ◽

John F. Marko ◽

Keir C. Neuman

Keyword(s):

Dna Sequence ◽

Base Pair ◽

Dna Supercoiling ◽

Magnetic Tweezers ◽

Relative Probability ◽

Base Pairs ◽

Damage Sensing ◽

Mismatched Base Pair ◽

Monovalent Salt

We present a method of detecting sequence defects by supercoiling DNA with magnetic tweezers. The method is sensitive to a single mismatched base pair in a DNA sequence of several thousand base pairs. We systematically compare DNA molecules with 0 to 16 adjacent mismatches at 1 M monovalent salt and 3.5 pN force and show that, under these conditions, a single plectoneme forms and is stably pinned at the defect. We use these measurements to estimate the energy and degree of end-loop kinking at defects. From this, we calculate the relative probability of plectoneme pinning at the mismatch under physiologically relevant conditions. Based on this estimate, we propose that DNA supercoiling could contribute to mismatch and damage sensing in vivo.

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DNA SEQUENCES OF FRAMESHIFT AND OTHER MUTATIONS INDUCED BY ICR-170 IN YEAST

Genetics ◽

10.1093/genetics/111.2.233 ◽

1985 ◽

Vol 111 (2) ◽

pp. 233-241

Author(s):

Joachim F Ernst ◽

D Michael Hampsey ◽

Fred Sherman

Keyword(s):

Saccharomyces Cerevisiae ◽

Amino Acid ◽

Sequence Analysis ◽

Genetic Analysis ◽

Dna Sequences ◽

Induced Mutations ◽

Sequence Analyses ◽

Base Pairs ◽

Yeast Saccharomyces Cerevisiae ◽

Frameshift Mutations

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.

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Repetitive Dictyostelium heat-shock promotor functions in Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.4.4.591 ◽

1984 ◽

Vol 4 (4) ◽

pp. 591-598 ◽

Cited By ~ 10

Author(s):

J Cappello ◽

C Zuker ◽

H F Lodish

Keyword(s):

Saccharomyces Cerevisiae ◽

Heat Shock ◽

Nucleotide Sequence ◽

Dna Sequence ◽

Base Pair ◽

Shock Treatment ◽

Yeast Cells ◽

Genomic Segment ◽

Heat Shock Treatment ◽

Shock Response

The Dictyostelium genome contains 40 copies of a 4.7-kilobase repetitive and apparently transposable DNA sequence (DIRS-1) and about 250 smaller elements that appear to be deletions or rearrangements of DIRS-1. Transcripts of these sequences are induced during differentiation and also by heat shock treatment of growing cells. We showed that one such cloned element, pB41.6 (2.5 kilobases) contains a nucleotide sequence identical to the Drosophila consensus heat shock promotor. To test whether this sequence might indeed control the expression of DIRS-1-related RNAs, we have cloned this genomic segment into yeast cells. In yeast cells, 41.6 directs synthesis of a 1.7-kilobase RNA that is induced at least 10-fold by heat shock. Transcription initiates at about 124 bases 3' of the putative promotor sequence and terminates within the 41.6 insert. A 381-base-pair subclone that contains the putative promotor sequence is sufficient to induce the heat shock response of 41.6 in yeast cells.

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