scholarly journals Identification of a Mutant DNA Polymerase δ in Saccharomyces cerevisiae With an Antimutator Phenotype for Frameshift Mutations

Genetics ◽  
2001 ◽  
Vol 158 (1) ◽  
pp. 177-186 ◽  
Author(s):  
Michalis I Hadjimarcou ◽  
Robert J Kokoska ◽  
Thomas D Petes ◽  
Linda J Reha-Krantz
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Polymerase ◽  
Bacteriophage T4 ◽  
Critical Role ◽  
Site Directed Mutagenesis ◽  
Base Substitution ◽  
Msh2 Gene ◽  
Frameshift Mutations ◽  
Frameshift Mutagenesis

Abstract We propose that a β-turn-β structure, which plays a critical role in exonucleolytic proofreading in the bacteriophage T4 DNA polymerase, is also present in the Saccharomyces cerevisiae DNA pol δ. Site-directed mutagenesis was used to test this proposal by introducing a mutation into the yeast POL3 gene in the region that encodes the putative β-turn-β structure. The mutant DNA pol δ has a serine substitution in place of glycine at position 447. DNA replication fidelity of the G447S-DNA pol δ was determined in vivo by using reversion and forward assays. An antimutator phenotype for frameshift mutations in short homopolymeric tracts was observed for the G447S-DNA pol δ in the absence of postreplication mismatch repair, which was produced by inactivation of the MSH2 gene. Because the G447S substitution reduced frameshift but not base substitution mutagenesis, some aspect of DNA polymerase proofreading appears to contribute to production of frameshifts. Possible roles of DNA polymerase proofreading in frameshift mutagenesis are discussed.

A MAJOR ROLE FOR BACTERIOPHAGE T4 DNA POLYMERASE IN FRAMESHIFT MUTAGENESIS

Genetics ◽  
1983 ◽  
Vol 103 (3) ◽  
pp. 353-366
Author(s):  
Lynn S Ripley ◽  
Nadja B Shoemaker
Keyword(s):  
Dna Polymerase ◽  
Dna Sequences ◽  
Frameshift Mutation ◽  
Bacteriophage T4 ◽  
Base Substitution ◽  
Temperature Sensitive ◽  
Polymerase Gene ◽  
Frameshift Mutagenesis ◽  
Substitution Mutations ◽  
Polymerase Mutants

ABSTRACT T4 DNA polymerase strongly influences the frequency and specificity of frameshift mutagenesis. Fifteen of 19 temperature-sensitive alleles of the DNA polymerase gene substantially influenced the reversion frequencies of frameshift mutations measured in the T4 rII genes. Most polymerase mutants increased frameshift frequencies, but a few alleles (previously noted as antimutators for base substitution mutations) decreased the frequencies of certain frameshifts while increasing the frequencies of others. The various patterns of enhanced or decreased frameshift mutation frequencies suggest that T4 DNA polymerase is likely to play a variety of roles in the metabolic events leading to frameshift mutation. A detailed genetic study of the specificity of the mutator properties of three DNA polymerase alleles (tsL56, tsL98 and tsL88) demonstrated that each produces a distinctive frameshift spectrum. Differences in frameshift frequencies at similar DNA sequences within the rII genes, the influence of mutant polymerase alleles on these frequencies, and the presence or absence of the dinucleotide sequence associated with initiation of Okazaki pieces at the frameshift site has led us to suggest that the discontinuities associated with discontinuous DNA replication may contribute to spontaneous frameshift mutation frequencies in T4.

scholarly journals The specificity of topoisomerase-mediated DNA cleavage defines acridine-induced frameshift specificity within a hotspot in bacteriophage T4.

Genetics ◽  
1991 ◽  
Vol 127 (3) ◽  
pp. 453-462 ◽  
Author(s):  
M Masurekar ◽  
K N Kreuzer ◽  
L S Ripley
Keyword(s):  
Dna Sequence ◽  
Dna Cleavage ◽  
Frameshift Mutation ◽  
Bacteriophage T4 ◽  
Precise Location ◽  
Frameshift Mutations ◽  
Phosphodiester Bond ◽  
Frameshift Mutagenesis

Abstract Acridine-induced frameshift mutations in bacteriophage T4 occur at the precise location in the DNA at which acridines stimulate DNA cleavage by the T4-encoded type II topoisomerase in vitro. The mutations are duplications or deletions that begin precisely at the broken phosphodiester bond. In vivo, acridine-induced frameshift mutagenesis is reduced nearly to background levels when the topoisomerase is genetically inactivated. These observations are consistent with a model in which cleaved DNA, induced by the topoisomerase and acridine, serves as the substrate for the production of frameshift mutations at the same site. Our model predicts that the specificity and frequency of cleavage direct the specificity and frequency of mutagenesis. This prediction was tested by examining the influence of DNA sequence changes on topoisomerase-mediated cleavage and on mutagenesis in the T4 rIIB gene. The model successfully predicted the results. When DNA sequence changes altered the position of acridine-induced, topoisomerase-mediated DNA cleavage in vitro, frameshift mutations were found at the new positions. DNA sequence changes that strongly decreased in vitro cleavage also reduced mutagenesis at that site. These results demonstrate that acridine-induced frameshift mutation specificity is directed by the characteristics of the acridine-topoisomerase reaction and do not suggest that slipped pairing in repeated sequences plays a major role in acridine-induced frameshifts in bacteriophage T4.

scholarly journals Mutational analysis of the mRNA operator for T4 DNA polymerase.

Genetics ◽  
1991 ◽  
Vol 128 (2) ◽  
pp. 203-213 ◽  
Author(s):  
M D Andrake ◽  
J D Karam
Keyword(s):  
Dna Polymerase ◽  
Mutational Analysis ◽  
Bacteriophage T4 ◽  
Spatial Arrangement ◽  
Loop Sequence ◽  
Shine Dalgarno Sequence ◽  
In Vitro Effects ◽  
Rna Hairpin

Abstract Biosynthesis of bacteriophage T4 DNA polymerase is autogenously regulated at the translational level. The enzyme, product of gene 43, represses its own translation by binding to its mRNA 5' to the initiator AUG at a 36-40 nucleotide segment that includes the Shine-Dalgarno sequence and a putative RNA hairpin structure consisting of a 5-base-pair stem and an 8-base loop. We constructed mutations that either disrupted the stem or altered specific loop residues of the hairpin and found that many of these mutations, including single-base changes in the loop sequence, diminished binding of purified T4 DNA polymerase to its RNA in vitro (as measured by a gel retardation assay) and derepressed synthesis of the enzyme in vivo (as measured in T4 infections and by recombinant-plasmid-mediated expression). In vitro effects, however, were not always congruent with in vivo effects. For example, stem pairing with a sequence other than wild-type resulted in normal protein binding in vitro but derepression of protein synthesis in vivo. Similarly, a C----A change in the loop had a small effect in vitro and a strong effect in vivo. In contrast, an A----U change near the base of the hairpin that was predicted to increase the length of the base-paired stem had small effects both in vitro and in vivo. The results suggest that interaction of T4 DNA polymerase with its structured RNA operator depends on the spatial arrangement of specific nucleotide residues and is subject to modulation in vivo.

Evidence that POB1, a Saccharomyces cerevisiae protein that binds to DNA polymerase alpha, acts in DNA metabolism in vivo

1992 ◽  
Vol 12 (12) ◽  
pp. 5724-5735
Author(s):  
J Miles ◽  
T Formosa
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
Dna Polymerase ◽  
Yeast Cells ◽  
Chromosome Loss ◽  
Dna Metabolism ◽  
Checkpoint Gene ◽  
Dna Polymerase Alpha ◽  
In Cells

Potential DNA replication accessory factors from the yeast Saccharomyces cerevisiae have previously been identified by their ability to bind to DNA polymerase alpha protein affinity matrices (J. Miles and T. Formosa, Proc. Natl. Acad. Sci. USA 89:1276-1280, 1992). We have now used genetic methods to characterize the gene encoding one of these DNA polymerase alpha-binding proteins (POB1) to determine whether it plays a role in DNA replication in vivo. We find that yeast cells lacking POB1 are viable but display a constellation of phenotypes indicating defective DNA metabolism. Populations of cells lacking POB1 accumulate abnormally high numbers of enlarged large-budded cells with a single nucleus at the neck of the bud. The average DNA content in a population of cells lacking POB1 is shifted toward the G2 value. These two phenotypes indicate that while the bulk of DNA replication is completed without POB1, mitosis is delayed. Deleting POB1 also causes elevated levels of both chromosome loss and genetic recombination, enhances the temperature sensitivity of cells with mutant DNA polymerase alpha genes, causes increased sensitivity to UV radiation in cells lacking a functional RAD9 checkpoint gene, and causes an increased probability of death in cells carrying a mutation in the MEC1 checkpoint gene. The sequence of the POB1 gene indicates that it is identical to the CTF4 (CHL15) gene identified previously in screens for mutations that diminish the fidelity of chromosome transmission. These phenotypes are consistent with defective DNA metabolism in cells lacking POB1 and strongly suggest that this DNA polymerase alpha-binding protein plays a role in accurately duplicating the genome in vivo.

Identification of pre-mRNA polyadenylation sites in Saccharomyces cerevisiae

1992 ◽  
Vol 12 (9) ◽  
pp. 4215-4229
Author(s):  
S Heidmann ◽  
B Obermaier ◽  
K Vogel ◽  
H Domdey
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Signal Sequence ◽  
Site Directed Mutagenesis ◽  
Yeast Cells ◽  
Yeast Saccharomyces Cerevisiae ◽  
Adh1 Gene ◽  
Polyadenylation Sites ◽  
Yeast Genes

In contrast to higher eukaryotes, little is known about the nature of the sequences which direct 3'-end formation of pre-mRNAs in the yeast Saccharomyces cerevisiae. The hexanucleotide AAUAAA, which is highly conserved and crucial in mammals, does not seem to have any functional importance for 3'-end formation in yeast cells. Instead, other elements have been proposed to serve as signal sequences. We performed a detailed investigation of the yeast ACT1, ADH1, CYC1, and YPT1 cDNAs, which showed that the polyadenylation sites used in vivo can be scattered over a region spanning up to 200 nucleotides. It therefore seems very unlikely that a single signal sequence is responsible for the selection of all these polyadenylation sites. Our study also showed that in the large majority of mRNAs, polyadenylation starts directly before or after an adenosine residue and that 3'-end formation of ADH1 transcripts occurs preferentially at the sequence PyAAA. Site-directed mutagenesis of these sites in the ADH1 gene suggested that this PyAAA sequence is essential for polyadenylation site selection both in vitro and in vivo. Furthermore, the 3'-terminal regions of the yeast genes investigated here are characterized by their capacity to act as signals for 3'-end formation in vivo in either orientation.

scholarly journals Analysis of Damage Tolerance Pathways in Saccharomyces cerevisiae: a Requirement for Rev3 DNA Polymerase in Translesion Synthesis

1998 ◽  
Vol 18 (2) ◽  
pp. 960-966 ◽  
Author(s):  
K. Baynton ◽  
A. Bresson-Roy ◽  
R. P. P. Fuchs
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Polymerase ◽  
Excision Repair ◽  
Translesion Synthesis ◽  
Hybridization Technique ◽  
Nucleotide Excision ◽  
Yeast Transformants ◽  
Plasmid Design ◽  
First Time

ABSTRACT The replication of double-stranded plasmids containing a singleN-2-acetylaminofluorene (AAF) adduct located in a short, heteroduplex sequence was analyzed in Saccharomyces cerevisiae. The strains used were proficient or deficient for the activity of DNA polymerase ζ (REV3 andrev3Δ, respectively) in a mismatch and nucleotide excision repair-defective background (msh2Δ rad10Δ). The plasmid design enabled the determination of the frequency with which translesion synthesis (TLS) and mechanisms avoiding the adduct by using the undamaged, complementary strand (damage avoidance mechanisms) are invoked to complete replication. To this end, a hybridization technique was implemented to probe plasmid DNA isolated from individual yeast transformants by using short, 32P-end-labeled oligonucleotides specific to each strand of the heteroduplex. In both the REV3 and rev3Δ strains, the two strands of an unmodified heteroduplex plasmid were replicated in ∼80% of the transformants, with the remaining 20% having possibly undergone prereplicative MSH2-independent mismatch repair. However, in the presence of the AAF adduct, TLS occurred in only 8% of theREV3 transformants, among which 97% was mostly error free and only 3% resulted in a mutation. All TLS observed in theREV3 strain was abolished in the rev3Δ mutant, providing for the first time in vivo biochemical evidence of a requirement for the Rev3 protein in TLS.

scholarly journals Pairing properties of bromouracil and repair of bromouracil-containing DNA. Possible utilization of bromodeoxyuridine triphosphate for site-directed mutagenesis

1988 ◽  
Vol 253 (3) ◽  
pp. 637-643 ◽  
Author(s):  
M Muller ◽  
J Martial ◽  
W G Verly
Keyword(s):  
Dna Polymerase ◽  
Site Directed Mutagenesis ◽  
Klenow Fragment ◽  
Single Experiment ◽  
E Coli ◽  
Partial Repair ◽  
Before And After ◽  
Polymerase I

5-Bromo-2′-deoxyuridine triphosphate (Br-dUTP) and dTTP are used interchangeably for DNA synthesis in vitro by the Klenow fragment of Escherichia coli DNA polymerase I. When DNA containing Br-dUMP instead of dTMP at a few preselected sites is transfected into competent bacteria, no mutation occurs, indicating that in vivo E. coli DNA polymerase always places a dAMP residue in front of any unrepaired Br-dUMP residue. On the other hand, in vitro Br-dUTP can also replace dCTP, but only with difficulty: when dCTP is absent, Br-dUMP can be forced in front of a dGMP residue, but the Klenow polymerase pauses before and after addition of Br-dUMP. Transfection into E. coli of the substituted DNA leads to the expected G→A transitions. These mutations can easily be targeted by using a suitable primer and the correctly chosen mix of deoxynucleoside triphosphates containing Br-dUTP. When Br-dUMP has been placed in front of a dGMP residue, the mutation yield is not 100%, showing a partial repair of the transfected DNA before it is replicated. Advantage can be taken of this partial repair to prepare a set of different mutations within a target region in a single experiment.

scholarly journals The B subunit of the DNA polymerase alpha-primase complex in Saccharomyces cerevisiae executes an essential function at the initial stage of DNA replication.

1994 ◽  
Vol 14 (2) ◽  
pp. 923-933 ◽  
Author(s):  
M Foiani ◽  
F Marini ◽  
D Gamba ◽  
G Lucchini ◽  
P Plevani
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
Cell Viability ◽  
Dna Polymerase ◽  
Chromosomal Dna ◽  
Dna Polymerase Alpha ◽  
B Subunit ◽  
Primase Complex

The four-subunit DNA polymerase alpha-primase complex is unique in its ability to synthesize DNA chains de novo, and some in vitro data suggest its involvement in initiation and elongation of chromosomal DNA replication, although direct in vivo evidence for a role in the initiation reaction is still lacking. The function of the B subunit of the complex is unknown, but the Saccharomyces cerevisiae POL12 gene, which encodes this protein, is essential for cell viability. We have produced different pol12 alleles by in vitro mutagenesis of the cloned gene. The in vivo analysis of our 18 pol12 alleles indicates that the conserved carboxy-terminal two-thirds of the protein contains regions that are essential for cell viability, while the more divergent NH2-terminal portion is partially dispensable. The characterization of the temperature-sensitive pol12-T9 mutant allele demonstrates that the B subunit is required for in vivo DNA synthesis and correct progression through S phase. Moreover, reciprocal shift experiments indicate that the POL12 gene product plays an essential role at the early stage of chromosomal DNA replication, before the hydroxyurea-sensitive step. A model for the role of the B subunit in initiation of DNA replication at an origin is presented.

scholarly journals DNA polymerase I is required for premeiotic DNA replication and sporulation but not for X-ray repair in Saccharomyces cerevisiae

1989 ◽  
Vol 9 (2) ◽  
pp. 365-376
Author(s):  
M E Budd ◽  
K D Wittrup ◽  
J E Bailey ◽  
J L Campbell
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
Dna Polymerase ◽  
Temperature Sensitive ◽  
Dna Polymerase I ◽  
X Ray ◽  
Temperature Sensitive Mutants ◽  
Polymerase I ◽  
Dna Polymerase Ii

We have used a set of seven temperature-sensitive mutants in the DNA polymerase I gene of Saccharomyces cerevisiae to investigate the role of DNA polymerase I in various aspects of DNA synthesis in vivo. Previously, we showed that DNA polymerase I is required for mitotic DNA replication. Here we extend our studies to several stages of meiosis and repair of X-ray-induced damage. We find that sporulation is blocked in all of the DNA polymerase temperature-sensitive mutants and that premeiotic DNA replication does not occur. Commitment to meiotic recombination is only 2% of wild-type levels. Thus, DNA polymerase I is essential for these steps. However, repair of X-ray-induced single-strand breaks is not defective in the DNA polymerase temperature-sensitive mutants, and DNA polymerase I is therefore not essential for repair of such lesions. These results suggest that DNA polymerase II or III or both, the two other nuclear yeast DNA polymerases for which roles have not yet been established, carry out repair in the absence of DNA polymerase I, but that DNA polymerase II and III cannot compensate for loss of DNA polymerase I in meiotic replication and recombination. These results do not, however, rule out essential roles for DNA polymerase II or III or both in addition to that for DNA polymerase I.

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