Suppressor analysis of temperature-sensitive RNA polymerase I mutations in Saccharomyces cerevisiae: suppression of mutations in a zinc-binding motif by transposed mutant genes.

Starting with two temperature-sensitive mutants (rpa190-1 and rpa190-5) of Saccharomyces cerevisiae, both of which are amino acid substitutions in the putative zinc-binding domain of the largest subunit (A190) of RNA polymerase I, we have isolated many independent pseudorevertants carrying extragenic suppressors (SRP) of rpa190 mutations. All the SRP mutations were dominant over the corresponding wild-type genes. They were classified into at least seven different loci by crossing each suppressed mutant with all of the other suppressed mutants and analyzing segregants. SRP mutations representing each of the seven loci were studied for their effects on other known rpa190 mutations. All of the SRP mutations were able to suppress both rpa190-1 and rpa190-5. In addition, one particular suppressor, SRP5, was found to suppress two other rpa190 mutations as well as an rpa190 deletion. Southern blot analysis combined with genetic crosses demonstrated that SRP5 maps to a region on chromosome XV loosely linked to rpa190 and represents a transposed mutant gene in two copies. Analysis of the A190 subunit by using anti-A190 antiserum indicated that the cellular concentration of A190 and hence of RNA polymerase I decreases in rpa190-1 mutants after a shift to 37 degrees C and that in the mutant strain carrying SRP5 this decrease is partially alleviated, presumably because of increased synthesis caused by increased gene dosage. These results suggest that the zinc-binding domain plays an important role in protein-protein interaction essential for the assembly and/or stability of the enzyme, regardless of whether it also participates directly in the interaction of the assembled enzyme with DNA.     

The C-terminal domain of dnaQ contains the polymerase binding site

The Escherichia coli dnaQ gene encodes the 3'-->5' exonucleolytic proofreading (epsilon) subunit of DNA polymerase III (Pol III). Genetic analysis of dnaQ mutants has suggested that epsilon might consist of two domains, an N-terminal domain containing the exonuclease and a C-terminal domain essential for binding the polymerase (alpha) subunit. We have created truncated forms of dnaQ resulting in epsilon subunits that contain either the N-terminal or the C-terminal domain. Using the yeast two-hybrid system, we analyzed the interactions of the single-domain epsilon subunits with the alpha and theta subunits of the Pol III core. The DnaQ991 protein, consisting of the N-terminal 186 amino acids, was defective in binding to the alpha subunit while retaining normal binding to the theta subunit. In contrast, the NDelta186 protein, consisting of the C-terminal 57 amino acids, exhibited normal binding to the alpha subunit but was defective in binding to the theta subunit. A strain carrying the dnaQ991 allele exhibited a strong, recessive mutator phenotype, as expected from a defective alpha binding mutant. The data are consistent with the existence of two functional domains in epsilon, with the C-terminal domain responsible for polymerase binding.     

Mutations influencing the frr gene coding for ribosome recycling factor (RRF)

A total of 52 null, six reversion, and five silent mutations of frr (the gene encoding for ribosome recycling factor (RRF)) of Escherichia coli are discussed along with 12 temperature-sensitive (ts) mutations and 14 intergenic suppressor strains of ts RRF. The null mutations were classified into six different categories. A computer-based secondary structure analysis showed three domains; domain A which has the N-terminal helix, domain B which contains coil, alpha-helix and beta-strand structure, and domain C which is a C-terminal helix. The ts mutations fell into domains A and C but not in domain B. More than a half of the null mutations fell into domain B while the silent mutations fell outside domain B. Substitution of Arg132 in domain C by other amino acids was observed among five independently isolated null mutants. It is suggested that domain B is important for maintaining the RRF structure, while the region including Arg132 is one of the active sites. A total of 14 intergenic suppressor strains of ts RRF were grouped into four categories, depending on which temperature-sensitive alleles were suppressed.     

Mutations at the putative junction sites of the yeast VMA1 protein, the catalytic subunit of the vacuolar membrane H(+)-ATPase, inhibit its processing by protein splicing.

A single gene, VMA1, encodes the 69-kDa subunit of the vacuolar membrane H(+)-ATPase in the yeast Saccharomyces cerevisiae. We have proposed that the subunit is synthesized as a precursor of 120 kDa (1,071 amino acids) and then converted to the 69-kDa form by an unusual processing reaction, which removes the internal domain of 454 amino acids (residues 284-737) and joins the N- and C-terminal domains. Cysteine to serine mutations at residues 284 and 738, the residues that bracket the internal domain, were introduced into the VMA1 gene by site-directed mutagenesis, and the mutant genes were expressed in a null vma1 mutant. Cells harboring either of the mutant vma1 genes accumulate nonfunctional fragments of the subunit. The mutation of Cys-284 inhibited the cleavage of the N-terminal junction site. Cys-738-->Ser mutation appeared to block the processing at both junction sites although the mutant gene yielded a small fraction of the functional 69-kDa subunit.     

RPA190, the gene coding for the largest subunit of yeast RNA polymerase A

Yeast RNA polymerases are being extensively studied at the gene level. The entire gene encoding the largest subunit of RNA polymerase A, A190, was isolated and characterized in detail. Southern hybridization and gene disruption experiments showed that the RPA190 gene is unique in the haploid yeast genome and essential for cell viability. Nuclease S1 mapping was used to identify mRNA 5' and 3' termini. RPA190 encodes a polypeptide chain of 186,270 daltons in a large uninterrupted reading frame. A dot matrix comparison of the deduced amino acid sequence of subunit A190 with Escherichia coli beta' and cognate subunits B220 and C160 from yeast RNA polymerases B and C showed a conserved pattern of homology regions (I-VI). A potential DNA-binding site (zinc-binding motif) is conserved in the N-terminal region I. Remarkably, the A190 subunit does not harbor the heptapeptide repeated sequence present in the B220 subunit. The sequence of the A190 subunit diverges from B220 and C160 by the presence of two hydrophilic domains inserted between homology regions I and II, and V and VI. From their codon usage and third base pyrimidine bias, RNA polymerase genes RPA190, RPB220, RPC160, and RPC40 fall among yeast genes expressed at an average level. The RPA190 5'-flanking region contains features present in other polymerase genes that might function in regulation.     

Mgs1 and Rad18/Rad5/Mms2 are required for survival of Saccharomyces cerevisiae mutants with novel temperature/cold sensitive alleles of the DNA polymerase delta subunit, Pol31

Saccharomyces cerevisiae DNA polymerase delta (Pol delta) is a heterotrimeric enzyme consisting of Pol3 (the catalytic subunit), Pol31 and Pol32. New pol31 alleles were constructed by introducing mutations into conserved amino acid residues in all 10 identified regions of Pol31. Six novel temperature-sensitive (ts) or cold-sensitive (cs) alleles, carrying mutations in regions III, IV, VII, VIII or IX, conferred a range of defects in the response to replication stress or DNA damage. Deletion of SGS1, RAD52, SRS2, MRC1 or RAD24 had a deleterious effect only in combination with those pol31 alleles that had a phenotype as single mutants, suggesting a requirement for recombination and checkpoint functions in processing the DNA lesions or structures that form as a consequence of replication with a defective Pol delta. In contrast, deletion of POL32 negatively affected the growth of almost all pol31 mutants, suggesting an important role for all conserved amino acids of Pol31 in maintaining the integrity of Pol delta complex structurally, at least in the absence of the third subunit. Surprisingly, deletions of RAD18 and MGS1 aggravated the temperature sensitivity conferred by most ts or cs alleles and specifically suppressed the hys2-1 and hys2-1-like mutations of POL31. Deletion of RAD5 or MMS2 had an effect on pol31 ts/cs mutants similar to that of RAD18, whereas deletion of RAD30 or REV3 had no effect. We propose that Rad18/Rad5/Mms2 and Mgs1 are required to promote replication when forks are destabilized or stalled due to defects in Pol delta. These data are consistent with the biochemical activity of the human Mgs1 orthologue, which binds and stimulates Pol deltain vitro. We also demonstrate that Mgs1 interacts physically with Pol31 in vivo. Moreover, regions I and VII of Pol31, which are specifically sensitive to high levels of Mgs1 and PCNA, could be sites of interaction.     

Yeast gene SRP1 (serine-rich protein). Intragenic repeat structure and identification of a family of SRP1-related DNA sequences

We have isolated and sequenced a yeast gene encoding a protein (Mr 24,875) very rich in serine (SRP) and alanine residues that accounted for 25% and 20% of the total amino acids, respectively. The SRP1 gene is highly expressed in culture conditions leading to glucose repression (Marguet & Lauquin, 1986), the amount of SRP1 mRNA representing about 1 to 2% of total poly(A)+ RNA. A repetitive structure of eight direct tandem repeats 36-base long, also reflected in the amino acid sequence, was found in the second half of the open reading frame. The consensus amino acid sequence of the repeat was Ser-Ser-Ser-Ala-Ala-Pro-Ser-Ser-Ser-Glu-Ala-Lys. Replacing the genomic copy of the cloned gene with a disrupted SRP1 gene indicated that the SRP1 gene was not essential for viability in yeast, but several SRP1-homologous sequences were found within the yeast genome, raising the possibility that the disrupted SRP1 gene is rescued by one of the other SRP-homologous sequences. Complete separation of yeast chromosomes by contour-clamped homogeneous field electrophoresis indicated that, apart from chromosome V, which carries the SRP1 gene, 12 chromosomes have SRP-related sequences with various degrees of homology. These sequences were located on chromosomes XV, VII and XI under stringent conditions of hybridization (tm -20 degrees C), and observed on chromosomes I, II, III, IV, VI, VIII, X, XI and XII, only under low-stringency conditions (tm -40 degrees C). Northern blot analysis of both the wild type and SRP1-disrupted strains indicated that along with SRP1 at least one more member of the SRP family was transcribed to a 0.7 kb (1 kb = 10(3) bases) polyadenylated RNA species clearly distinct from the SRP1-specific mRNA (1 kb long). Analyses of the SRP1 repeat domain suggested a model for the divergent evolution of the repeats in the SRP1 sequence.     

Isolation and characterization of temperature-sensitive and thermostable mutants of the human receptor-like protein tyrosine phosphatase LAR.

Human LAR is a transmembrane receptor-like protein whose cytoplasmic region contains two tandemly duplicated domains homologous to protein tyrosine phosphatases (PTPases). Whereas the membrane-proximal domain I has enzymatic activity, the membrane-distal domain II has no apparent catalytic activity but seems to have a regulatory function. In order to study structure-function relationships of the LAR PTPase, LAR domain I was expressed in Escherichia coli, and mutants that have reduced catalytic activity or reduced thermostability were isolated and characterized. We isolated 18 unique hydroxylamine-induced missense mutations in the LAR domain I segment, of which three were temperature-sensitive. Five additional temperature-sensitive mutations were isolated using N-methyl-N'-nitro-N-nitrosoguanidine. All eight temperature-sensitive mutations are confined within a short segment of the LAR domain I sequence between amino acid positions 1329 and 1407. To examine whether this region is particularly prone to temperature-sensitive mutations, tyrosine at amino acid position 1379 was changed to a phenylalanine by oligonucleotide-directed mutagenesis. This mutant, Y1379-F, was indeed temperature-sensitive. We also isolated a revertant of a temperature-sensitive mutant. The revertant contained a second-site mutation (C1446-Y) that suppresses several temperature-sensitive mutations and also enhances the folding of LAR protein produced in E. coli.     

The Pol32 subunit of DNA polymerase delta contains separable domains for processive replication and proliferating cell nuclear antigen (PCNA) binding

We have carried out a domain analysis of POL32, the third subunit of Saccharomyces cerevisiae DNA polymerase delta (Pol delta). Interactions with POL31, the second subunit of Pol delta, are specified by the amino-terminal 92 amino acids, whereas interactions with the replication clamp proliferating cell nuclear antigen (PCNA, POL30) reside at the extreme carboxyl-terminal region. Pol32 binding, in vivo and in vitro, to the large subunit of DNA polymerase alpha, POL1, requires the carboxyl-proximal region of Pol32. The amino-terminal region of Pol32 is essential for damage-induced mutagenesis. However, the presence of its carboxyl-terminal PCNA-binding domain enhances the efficiency of mutagenesis, particularly at high loads of DNA damage. In vitro, in the absence of effector DNA, the PCNA-binding domain of Pol32 is essential for PCNA-Pol delta interactions. However, this domain has minimal importance for processive DNA synthesis by the ternary DNA-PCNA-Pol delta complex. Rather, processivity is determined by PCNA-binding domains located in the Pol3 and/or Pol31 subunits. Using diagnostic PCNA mutants, we show that during DNA synthesis the carboxyl-terminal domain of Pol32 interacts with the carboxyl-terminal region of PCNA, whereas interactions of the other subunit(s) of Pol delta localize largely to a hydrophobic pocket at the interdomain connector loop region of PCNA.     

Rpa12p, a conserved RNA polymerase I subunit with two functional domains

Rpa12p is a subunit of RNA polymerase I formed of two zinc-binding domains. The N-terminal zinc region (positions 1-60) is poorly conserved from yeast to man. The C-terminal domain contains an invariant Q.RSADE.T.F motif shared with the TFIIS elongation factor of RNA polymerase II and its archaeal counterpart. Deletions removing the N-terminal domain fail to grow at 34 degrees C, are sensitive to nucleotide-depleting drugs and become lethal in rpa14-Delta mutants lacking the non-essential RNA polymerase I subunit Rpa14p. They also strongly alter the immunofluorescent properties of RNA polymerase I in the nucleolus. Finally, they prevent the binding of Rpa12p to immunopurified polymerase I and impair a specific two-hybrid interaction with the second largest subunit. In all these respects, N-terminal deletions behave like full deletions. In contrast, C-terminal deletions retaining only the first N-terminal 60 amino acids are indistinguishable from wild type. Thus, the N-terminal zinc domain of Rpa12p determines its anchoring to RNA polymerase I and is the only critical part of that subunit in vivo.     

Structure of the genes encoding the rod-core linker polypeptides of Mastigocladus laminosus phycobilisomes and functional aspects of the phycobiliprotein/linker-polypeptide interactions.

The 3' portion of the cpc operon in Mastigocladus laminosus encloses the genes 5'-cpcF-cpcG1-cpcG2-cpcG3 3'. The three cpcG genes encode different phycocyanin-associated rod-core linker polypeptides of the phycobilisomes with predicted 279, 247 and 254 amino acids in length. The gene products CpcG show a high similarity at their N-terminal domains (190 amino acids) and an overall identity of 47-53% to one another. Each of the three CpcG polypeptides is highly related to one of the four CpcG gene products of Anabaena sp. PCC 7120 (66-81% identity). It is suggested that these pairs of rod-core linker polypeptides mediate the same specific type of phycocyanin----allophycocyanin interaction in the similar phycobilisomes of M. laminosus and Anabaena sp. PCC 7120. The similarity of the CpcG1, CpcG2 and CpcG3 polypeptides to the single CpcG rod-core linker polypeptide of Synechococcus sp. PCC 7002 (36-41% identity) is lower. The rod-core linker polypeptides are more distantly related to the rod linker polypeptides associated with phycocyanin or phycoerythrin. However, six conserved domains were identified within the N-terminal 190 amino acids of these linker proteins, which bear similar amino acid sequences, including highly conserved basic amino acids. A similar amino acid sequence but with conserved acidic amino acids can be found in the beta subunits of phycocyanin, phycoerythrin and phycoerythrocyanin, which is protruding into the central cavity of the phycobiliprotein hexamers. It is suggested that these domains are sites of phycobiliprotein-hexamer/rod and rod-core linker interactions.     

Mutator mutants of Escherichia coli carrying a defect in the DNA polymerase III tau subunit

To investigate the possible role of accessory subunits of Escherichia coli DNA polymerase III holoenzyme (HE) in determining chromosomal replication fidelity, we have investigated the role of the dnaX gene. This gene encodes both the tau and gamma subunits of HE, which play a central role in the organization and functioning of HE at the replication fork. We find that a classical, temperature-sensitive dnaX allele, dnaX36, displays a pronounced mutator effect, characterized by an unusual specificity: preferential enhancement of transversions and -1 frameshifts. The latter occur predominantly at non-run sequences. The dnaX36 defect does not affect the gamma subunit, but produces a tau subunit carrying a missense substitution (E601K) in its C-terminal domain (domain V) that is involved in interaction with the Pol III alpha subunit. A search for new mutators in the dnaX region of the chromosome yielded six additional dnaX mutators, all carrying a specific tau subunit defect. The new mutators displayed phenotypes similar to dnaX36: strong enhancement of transversions and frameshifts and only weak enhancement for transitions. The combined findings suggest that the tau subunit of HE plays an important role in determining the fidelity of the chromosomal replication, specifically in the avoidance of transversions and frameshift mutations.