Characterization of the five replication factor C genes of Saccharomyces cerevisiae.

Replication factor C (RFC) is a five-subunit DNA polymerase accessory protein that functions as a structure-specific, DNA-dependent ATPase. The ATPase function of RFC is activated by proliferating cell nuclear antigen. RFC was originally purified from human cells on the basis of its requirement for simian virus 40 DNA replication in vitro. A functionally homologous protein complex from Saccharomyces cerevisiae, called ScRFC, has been identified. Here we report the cloning, by either peptide sequencing or by sequence similarity to the human cDNAs, of the S. cerevisiae genes RFC1, RFC2, RFC3, RFC4, and RFC5. The amino acid sequences are highly similar to the sequences of the homologous human RFC 140-, 37-, 36-, 40-, and 38-kDa subunits, respectively, and also show amino acid sequence similarity to functionally homologous proteins from Escherichia coli and the phage T4 replication apparatus. All five subunits show conserved regions characteristic of ATP/GTP-binding proteins and also have a significant degree of similarity among each other. We have identified eight segments of conserved amino acid sequences that define a family of related proteins. Despite their high degree of sequence similarity, all five RFC genes are essential for cell proliferation in S. cerevisiae. RFC1 is identical to CDC44, a gene identified as a cell division cycle gene encoding a protein involved in DNA metabolism. CDC44/RFC1 is known to interact genetically with the gene encoding proliferating cell nuclear antigen, confirming previous biochemical evidence of their functional interaction in DNA replication.

Identification of replication factor C from Saccharomyces cerevisiae: a component of the leading-strand DNA replication complex.

A number of proteins have been isolated from human cells on the basis of their ability to support DNA replication in vitro of the simian virus 40 (SV40) origin of DNA replication. One such protein, replication factor C (RFC), functions with the proliferating cell nuclear antigen (PCNA), replication protein A (RPA), and DNA polymerase delta to synthesize the leading strand at a replication fork. To determine whether these proteins perform similar roles during replication of DNA from origins in cellular chromosomes, we have begun to characterize functionally homologous proteins from the yeast Saccharomyces cerevisiae. RFC from S. cerevisiae was purified by its ability to stimulate yeast DNA polymerase delta on a primed single-stranded DNA template in the presence of yeast PCNA and RPA. Like its human-cell counterpart, RFC from S. cerevisiae (scRFC) has an associated DNA-activated ATPase activity as well as a primer-template, structure-specific DNA binding activity. By analogy with the phage T4 and SV40 DNA replication in vitro systems, the yeast RFC, PCNA, RPA, and DNA polymerase delta activities function together as a leading-strand DNA replication complex. Now that RFC from S. cerevisiae has been purified, all seven cellular factors previously shown to be required for SV40 DNA replication in vitro have been identified in S. cerevisiae.

Expression of the F glycoprotein gene from human respiratory syncytial virus in Escherichia coli: mapping of a fusion inhibiting epitope.

A cDNA copy of the gene encoding the entire amino acid sequence of the fusion (F) protein of human respiratory syncytial virus (strain A2) was inserted into a bacterial expression vector containing the lambda PR promoter. Upon heat induction, Escherichia coli cells harboring the vector produced a 45-kDa peptide which reacted with rabbit polyclonal antiserum to the native F protein. Expression of the F gene resulted in severe inhibition of bacterial growth, which was overcome by deletion of the DNA sequences encoding the F signal peptide. The region of the F protein which reacted with a virus-neutralizing and fusion-inhibiting monoclonal antibody was probed by expressing cDNA fragments encoding different protein domains in E. coli and testing antibody reactivity by Western blot analysis. Analysis of six fragments yielded an overlapping antibody-reactive region between amino acids 253 and 298. Analysis of reactivity with a cassette of synthetic peptides confirmed that the virus-neutralizing epitope mapped between residues 289 and 298 defined by the amino acid sequence M-S-I-I-K-E-E-V-L-A.

Alkaline phosphatase fusions to the respiratory syncytial virus F protein as an approach to analyze its membrane topology.

Manoil and Beckwith (1985) have constructed a transposon, TnphoA, that permits the generation of hybrid proteins composed of alkaline phosphatase (AP) lacking its signal peptide fused to amino-terminal sequences of other proteins. This transposon has been used to localize export signals and analyze membrane topology of bacterial proteins. We have applied this approach to the membrane fusion protein (F) of respiratory syncytial virus (RSV). The transposon TnphoA and a plasmid directing bacterial expression of the F gene were used to construct F-AP hybrids. These hybrids yielded AP activity, indicating the presence of viral sequences that promoted protein transport through the cytoplasmic membrane. Sequence analysis showed that TnphoA was inserted at four different positions within the F1 subunit. Deletion of the hydrophobic F1 amino-terminus (fusion-related domain) resulted in AP transport to the periplasm, suggesting that the hydrophobic amino-terminus of the F2 subunit is sufficient to promote protein export. Some hybrids were apparently cleaved at or near the F2/F1 junction. The periplasmic localization of an uncleaved hybrid strongly suggested that the fusion-related domain of the F protein, when in the uncleaved F0 precursor, can be moved across the bacterial cytoplasmic membrane. Although these results apply to the recombinant F protein, they agree with the presumed signal sequence and membrane topology of the native F glycoprotein. Thus, this method may be useful in determining membrane topology and in localizing important domains of viral proteins.

Mutations that alter the DNA binding site for the bacteriophage lambda cII protein and affect the translation efficiency of the cII gene.

The efficiency of translation of the cII gene of bacteriophage lambda is greatly reduced by the cII3059 mutation, a GUU----GAU (Val----Asp) change in the second cII codon. Mutations in the third and fourth codons of the cII gene, called ctr mutations, reverse this translation deficiency. Lambda cII3059 ctr-1, which has a GCA----ACA (Ala----Thr) change in the fourth cII codon, produces about half the normal level of cII activity in liquid cultures, and lambda cII3059 ctr-2 and lambda cII3059 ctr-3, which have identical CGT----CGC changes in the third codon, produce normal levels of cII activity in liquid culture. Since the cII protein of ctr-3 has the same primary sequence as that of lambda cII3059, the cII- phenotype of lambda cII3059 can be explained entirely by the deficiency of translating cII mRNA. We propose that ctr mutations increase translation efficiency by destabilizing a stable stem structure which can be formed by cII mRNA. The ctr mutations lie in an overlapping regulatory region which contains, in addition to sequence elements that influence the rate of cII translation, a region to which cII protein binds to activate transcription from the PRE promoter. The ctr-1 mutation alters the cII recognition sequence from 5'-T-T-G-C-N6T-T-G-C-3' to 5'-T-T-G-C-N6T-T-G-T-3', but has no effect on PRE activity. Since a C----T change in the first (5'-proximal) T-T-G-C sequence (to yield 5'-T-T-G-T-N6T-T-G-C) greatly lowers cII binding affinity, cII protein must not recognize the two T-T-G-C sequences in an identical manner.

CII-dependent activation of the pRE promoter of coliphage lambda fused to the Escherichia coli galK gene.

Using a cloning vector designed for the study of prokaryotic promoters by fusion to the Escherichia coli galactokinase gene (galK), we have constructed a plasmid in which the lambda pRE promoter controls galactokinase expression. A galK- host containing this plasmid has a Gal- phenotype since transcription from pRE requires activation by the lambda CII protein. When CII protein is provided by a prophage, galactokinase is synthesized at a rate dependent on the concentration of CII protein. A second plasmid was constructed in which the pRE promoter from phage 21 controls galactokinase expression. Transcription of the galK gene in this plasmid requires the phage 21 CII protein. Using this system, we demonstrate that the lambda and 21 pRE promoters are highly selective for their corresponding CII proteins. However, a cross-reaction between 21 pRE and the lambda CII protein was observed. In addition, we transferred the pRE-galK fusion unit from the plasmid to a phage, and then to the host chromosome in single copy. Galactokinase expression in this single copy pRE-galK system is also dependent on CII protein, which may be provided from a multicopy plasmid. The high concentration of CII protein provided by the plasmid results in maximal expression of the pRE-galK transcription unit. In this second system low levels of CII activity from CII- mutants are amplified and can be readily detected.