Phosphorylation of the RNA polymerase II carboxy-terminal domain by the Bur1 cyclin-dependent kinase.

BUR1, which was previously identified by a selection for mutations that have general effects on transcription in Saccharomyces cerevisiae, encodes a cyclin-dependent kinase that is essential for viability, but none of its substrates have been identified to date. Using an unbiased biochemical approach, we have identified the carboxy-terminal domain (CTD) of Rpb1, the largest subunit of RNA polymerase II, as a Bur1 substrate. Phosphorylation of Rpb1 by Bur1 is likely to be physiologically relevant, since bur1 mutations interact genetically with rpb1 CTD truncations and with mutations in other genes involved in CTD function. Several genetic interactions are presented, implying a role for Bur1 during transcriptional elongation. These results identify Bur1 as a fourth S. cerevisiae CTD kinase and provide striking functional similarities between Bur1 and metazoan P-TEFb.

BUR1 and BUR2 encode a divergent cyclin-dependent kinase-cyclin complex important for transcription in vivo.

BUR1 and BUR2 were previously identified by a genetic selection for mutations that increase transcription from basal promoters in vivo. BUR1 encoded a putative protein kinase with greatest similarity to members of the cyclin-dependent kinase (CDK) family, although that similarity was not sufficient to classify it as a CDK. It was also not known whether Bur1 activity was cyclin dependent and, if so, which cyclins stimulated Bur1. The molecular cloning and characterization of BUR2 presented here sheds light on these issues. Genetic analysis indicates that BUR2 function is intimately related to that of BUR1: bur1 and bur2 mutations cause nearly identical spectra of mutant phenotypes, and overexpression of BUR1 suppresses a bur2 null allele. Biochemical analysis has provided a molecular basis for these genetic observations. We find that BUR2 encodes a cyclin for the Bur1 protein kinase, based on the following evidence. First, the BUR2 amino acid sequence reveals similarity to the cyclins; second, Bur1 and Bur2 coimmunoprecipitate from crude extracts and interact in the two-hybrid system; and third, BUR2 is required for Bur1 kinase activity in vitro. Our combined genetic and biochemical results therefore indicate that Bur1 and Bur2 comprise a divergent CDK-cyclin complex that has an important functional role during transcription in vivo.

A new regulatory domain on the TATA-binding protein.

Recognition of the TATA box by the TATA-binding protein (TBP) is a highly regulated step in RNA polymerase II-dependent transcription. Several proteins have been proposed to regulate TBP activity, yet the TBP domains responsive to all these regulators have not been defined. Here we describe a new class of TBP mutants that increase transcription from core promoters in vivo. The majority of these mutations alter amino acids that cluster tightly on the TBP surface, defining a new TBP regulatory domain. The mutant TBP proteins are defective for binding the transcriptional regulator yNC2, are resistant to inhibition by yNC2 in vitro and exhibit allele-specific genetic interactions with yNC2. These results provide strong biochemical and genetic evidence that TBP is directly repressed in vivo, and define a new TBP domain important for transcriptional regulation.

Suppression mechanisms: themes from variations.

Suppressor analysis is a commonly used strategy to identify functional relationships between genes that might not have been revealed through other genetic or biochemical means. Many mechanisms that explain the phenomenon of genetic suppression have been described, but the wide variety of possible mechanisms can present a challenge to defining the relationship between a suppressor and the original gene. This article provides a broad framework for classifying suppression mechanisms and describes a series of genetic tests that can be applied to determine the most likely mechanism of suppression.

Saccharomyces cerevisiae BUR6 encodes a DRAP1/NC2alpha homolog that has both positive and negative roles in transcription in vivo.

BUR3 and BUR6 were identified previously by selecting for mutations that increase transcription from an upstream activating sequence (UAS)-less promoter in Saccharomyces cerevisiae. The bur3-1 and bur6-1 mutations are recessive, increase transcription from a suc2 delta uas allele, and cause other mutant phenotypes, suggesting that Bur3p and Bur6p function as general repressors of the basal transcriptional machinery. The molecular cloning and characterization of BUR3 and BUR6 are presented here. BUR3 is identical to MOT1, a previously characterized essential gene that encodes an ATP-dependent inhibitor of the TATA box-binding protein. Cloning and nucleotide sequence analysis reveals that BUR6 encodes a homolog of DRAP1 (also called NC2alpha), a mammalian repressor of basal transcription. Strains that contain a bur6 null allele are viable but grow extremely poorly, demonstrating that BUR6 is critical for normal cell growth in yeast. The Bur6p histone fold domain is required for function; an extensive nonoverlapping set of deletion alleles throughout the histone fold domain impairs BUR6 function in vivo, whereas mutations in the amino- and carboxy-terminal tails have no detectable effect. BUR6 and BUR3/MOT1 have different functions depending on promoter context: although the bur3-1 and bur6-1 mutations increase transcription from delta uas promoters, they result in reduced transcription from the wild-type GAL1 and GAL10 promoters. This transcriptional defect is due to the inability of the GAL10 UAS to function in bur6-1 strains. The similar phenotypes of bur6 and bur3 (mot1) mutations suggest that Bur6p and Mot1p have related, but not identical, functions in modulating the activity of the general transcription machinery in vivo.

Mutations that suppress the deletion of an upstream activating sequence in yeast: involvement of a protein kinase and histone H3 in repressing transcription in vivo.

Regulated transcription of most protein-encoding genes in Saccharomyces cerevisiae requires an upstream activating sequence (UAS); in the absence of UAS elements, little or no transcription occurs. In certain mutant strains, however, promoters that have been deleted for their UAS can direct significant levels of transcription, indicating that the remaining promoter elements (the basal promoter) are capable of directing higher levels of transcription, but they are normally represented in wild-type strains. To analyze this repression, we have selected for mutations that cause increased transcription of the SUC2 gene in the absence of its UAS. In addition to some previously studied genes, this selection has identified five genes that we have designated BUR1, BUR2, BUR3, BUR5 and BUR6 (for Bypass UAS Requirement). The bur mutations cause pleiotropic phenotypes, indicating that they affect transcription of many genes. Furthermore, some bur mutations suppress the requirement for the SNF5 trans-activator at both SUC2 and Ty. Additional analysis has demonstrated that BUR1 is identical to SGV1, which encodes a CDC28-related protein kinase. This result indicates that protein phosphorylation is important for repression of the SUC2 basal promoter as well as other aspects of transcription in vivo. Finally, BUR5 is identical to HHT1, encoding histone H3, further implicating chromatin structure as important for expression of SUC2.

Drosophila proliferating cell nuclear antigen. Structural and functional homology with its mammalian counterpart.

A protein with an apparent mass of 36 kDa was purified from Drosophila melanogaster embryos using a protocol developed for the purification of proliferating cell nuclear antigen (PCNA) from human 293 cells. The Drosophila protein comigrated with human PCNA on one-dimensional sodium dodecyl sulfate-polyacrylamide gels and cross-reacted with monoclonal anti-rabbit PCNA antibodies. NH2-terminal amino acid sequence analysis revealed that the putative Drosophila PCNA was highly homologous to human PCNA. Of the first 22 amino acids, 16 were identical, and 4 of the remaining 6 were changed conservatively. Results of total amino acid analysis were also consistent with a high degree of similarity between Drosophila PCNA and human PCNA. Functional analysis using the reconstituted simian virus 40 in vitro DNA replication system demonstrated that Drosophila PCNA could substitute, albeit with reduced efficiency, for human PCNA in stimulating simian virus 40 DNA synthesis. Affinity-purified anti-Drosophila PCNA antibodies cross-reacted with human PCNA and were able to recognize specifically Drosophila PCNA both on crude homogenate immunoblots and by indirect immunofluorescence analysis of proliferating cells in larval tissues in situ. These antibodies thus promise to be useful probes for the study of cell proliferation in this rapidly developing organism.

Replication of SV40 in vitro using proteins derived from a human cell extract.

In the presence of large T antigen and plasmids containing a functional origin of replication, extracts from a human cell line will support multiple rounds of simian virus 40 (SV40) replication in vitro. Fractionation of this extract has led to the identification of several factors, some of which have been purified to homogeneity. The characterisation of these proteins has led to the separation of SV40 replication in vitro into multiple stages. Two proteins, the cell cycle-regulated proliferating cell nuclear antigen and replication factor-C, have been shown to be essential for coordinating leading and lagging strand synthesis in this system. Another protein, replication factor-A, is a multi-subunit protein of 70, 34 and 11K (K = 10(3) Mr) polypeptides which, because of its high affinity for DNA, is thought to function as a eukaryotic single-stranded DNA binding protein. Interactions between other cellular factors are also described that effect the initiation of DNA replication, but are not required in a more purified system. In addition a model for a hypothetical replication fork is described, which suggests a role for both alpha- and delta-polymerases in this system, and may be applicable to higher eukaryotes.

Identification of cellular components required for SV40 DNA replication in vitro.

To investigate the cellular proteins involved in simian virus 40 (SV40) replication, extracts derived from human 293 cells have been fractionated into multiple components. When such fractions are combined with the virus-encoded T antigen (TAg) and SV40 origin containing plasmid DNA, efficient and complete replication is achieved, while each fraction alone is inactive. At present, a minimum of eight such cellular components have been identified. Previous experiments have demonstrated one of these to be the cell-cycle-regulated proliferating-cell nuclear antigen (PCNA). As PCNA has been identified as a processivity factor for DNA polymerase delta, we suggest that both polymerases alpha and delta are involved in this system. Three further fractions have been identified. One is a partially purified fraction which, under certain conditions, is required with TAg for the formation of a pre-synthesis complex of proteins at the replication origin. The second of these factors, RF-A, is a complex of three polypeptides which may function as a eucaryotic SSB. The third, RF-C, is a factor which is required, with PCNA, for coordinated leading- and lagging-strand synthesis at the replication fork. Complete synthesis and segregation of the daughter molecules also requires the presence of topoisomerases I and II. These results suggest a model for DNA synthesis which involves multiple stages prior to and during replicative DNA synthesis.

Coordinated leading and lagging strand synthesis during SV40 DNA replication in vitro requires PCNA.

Proliferating cell nuclear antigen (PCNA) is a cell cycle and growth regulated protein required for replication of SV40 DNA in vitro. Its function was investigated by comparison of the replication products synthesized in its presence or absence. In the completely reconstituted replication system that contains PCNA, DNA synthesis initiates at the origin and proceeds bidirectionally on both leading and lagging strands around the template DNA to yield duplex, circular daughter molecules. In contrast, in the absence of PCNA, early replicative intermediates containing short nascent strands accumulate. Replication forks continue bidirectionally from the origin, but surprisingly, only lagging strand products are synthesized. Thus two stages of DNA synthesis have been defined, with the second stage requiring PCNA for coordinated leading and lagging strand synthesis at the replication fork. We suggest that during eukaryotic chromosome replication there is a switch to a PCNA-dependent elongation stage that requires two distinct DNA polymerases.

Identification of multiple cellular factors required for SV40 replication in vitro.

The replication of simian virus 40 has been studied by using cell-free extracts derived from human 293 cells. Fractionation of this extract has led to the identification of three fractions that are required for efficient DNA synthesis. Initial fractionation of the crude extract by phosphocellulose chromatography has produced two fractions, I and II, neither of which is able to support replication separately, but when they are combined, efficient synthesis is restored. Both fractions are required, with SV40 T antigen, for the formation of a presynthesis complex at the SV40 origin. The major replication enzymes, DNA polymerase, DNA primase and the topoisomerases I and II all reside in fraction II. Fraction I has been subdivided into two subfractions (A and B) by DEAE-cellulose chromatography. Fraction A is essential for replication and is required for presynthesis complex formation. Fraction B stimulates DNA replication and is only required at the elongation stage. This multicomponent system has provided the foundation for identification of individual components that are required for DNA replication in vitro.

The cell-cycle regulated proliferating cell nuclear antigen is required for SV40 DNA replication in vitro.

Cell-free extracts prepared from human 293 cells, supplemented with purified SV40 large-T antigen, support replication of plasmids containing the SV40 origin of DNA replication. A cellular protein (Mr approximately 36,000) that is required for efficient SV40 DNA synthesis in vitro has been purified from these extracts. This protein is recognized by human autoantibodies and is identified as the cell-cycle regulated protein known as proliferating cell nuclear antigen (PCNA) or cyclin.

Functional identity of proliferating cell nuclear antigen and a DNA polymerase-delta auxiliary protein.

The mechanism of replication of the simian virus 40 (SV40) genome closely resembles that of cellular chromosomes, thereby providing an excellent model system for examining the enzymatic requirements for DNA replication. Only one viral gene product, the large tumour antigen (large-T antigen), is required for viral replication, so the majority of replication enzymes must be cellular. Indeed, a number of enzymatic activities associated with replication and the S phase of the cell cycle are induced upon SV40 infection. Cell-free extracts derived from human cells, when supplemented with immunopurified SV40 large-T antigen support efficient replication of plasmids that contain the SV40 origin of DNA replication. Using this system, a cellular protein of relative molecular mass 36,000 (Mr = 36K) that is required for the elongation stage of SV40 DNA replication in vitro has been purified and identified as a known cell-cycle regulated protein, alternatively called the proliferating cell nuclear antigen (PCNA) or cyclin. It was noticed that, in its physical characteristics, PCNA closely resembles a protein that regulates the activity of calf thymus DNA polymerase-delta. Here we show that PCNA and the polymerase-delta auxiliary protein have similar electrophoretic behaviour and are both recognized by anti-PCNA human autoantibodies. More importantly, both proteins are functionally equivalent; they stimulate SV40 DNA replication in vitro and increase the processivity of calf thymus DNA polymerase-delta. These results implicate a novel animal cell DNA polymerase, DNA polymerase-delta, in the elongation stage of replicative DNA synthesis in vitro.

Functional characterization of thermolabile DNA-binding proteins that affect adenovirus DNA replication.

The human adenovirus type 2 (Ad2) mutant Ad2ts111 has previously been shown to contain two mutations which result in a complex phenotype. Ad2ts111 contains a single base change in the early region 1B (E1B) 19,000-molecular-weight (19K) coding region which yields a cyt deg phenotype and another defect which maps to the E2A 72K DNA-binding protein (DBP) coding region that causes a temperature-sensitive DNA replication phenotype. Here we report that the defect in the Ad2ts111 DBP is due to a single G----T transversion that results in a substitution of valine for glycine at amino acid 280. A temperature-independent revertant, Ad2ts111R10, was isolated, which reverts back to glycine at amino acid 280 yet retains the cyt and deg phenotypes caused by the 19K mutation. We physically separated the two mutations of Ad2ts111 by constructing a recombinant virus, Ad2ts111A, which contained a wild-type Ad2 E1B 19K gene and the gly----val mutation in the 72K gene. Ad2ts111A was cyt+ deg+, yet it was still defective for DNA replication at the nonpermissive temperature. The Ad2ts111 DBP mutation is located only two amino acids away from the site of the mutation in Ad2+ND1ts23, a previously sequenced DBP mutant. Biochemical studies of purified Ad2+ND1ts23 DBP showed that this protein was defective for elongation but not initiation of replication in a cell-free replication system consisting of purified Ad polymerase, terminal protein precursor, and nuclear factor I. Ad2+ND1ts23 DBP bound less tightly to single-strand DNA than did Ad2 DBP, as shown by salt gradient elution of purified DBPs from denatured DNA cellulose columns. This decreased binding to DNA was probably due to local conformational changes in the protein at a site that is critical for DNA binding rather than to global changes in protein structure, since both the Ad2+ND1ts23 and Ad2 DBPs showed identical cleavage patterns by the protease thermolysin at various temperatures.