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.
The classical genetic approach for exploring biological pathways typically begins by identifying mutations that cause a phenotype of interest. Overexpression or misexpression of a wild-type gene product, however, can also cause mutant phenotypes, providing geneticists with an alternative yet powerful tool to identify pathway components that might remain undetected using traditional loss-of-function analysis. This review describes the history of overexpression, the mechanisms that are responsible for overexpression phenotypes, tests that begin to distinguish between those mechanisms, the varied ways in which overexpression is used, the methods and reagents available in several organisms, and the relevance of overexpression to human disease.
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.
The Bur1-Bur2 and Paf1 complexes function during transcription elongation and affect histone modifications. Here we describe new roles for Bur1-Bur2 and the Paf1 complex. We find that histone H3 K36 tri-methylation requires specific components of the Paf1 complex and that K36 trimethylation is more strongly affected at the 5 0 ends of genes in paf1D and bur2D strains in parallel with increased acetylation of histones H3 and H4. Interestingly, the 5 0 increase in histone acetylation is independent of K36 methylation, and therefore is mechanistically distinct from the methylation-driven deacetylation that occurs at the 3 0 ends of genes. Finally, Bur1-Bur2 and the Paf1 complex have a second methylation-independent function, since bur2D set2D and paf1D set2D double mutants display enhanced histone acetylation at the 3 0 ends of genes and increased cryptic transcription initiation. These findings identify new functions for the Paf1 and Bur1-Bur2 complexes, provide evidence that histone modifications at the 5 0 and 3 0 ends of coding regions are regulated by distinct mechanisms, and reveal that the Bur1-Bur2 and Paf1 complexes repress cryptic transcription through a Set2-independent pathway.
Modern genetic analysis requires the development of new resources to systematically explore gene function in vivo. Overexpression screens are a powerful method to investigate genetic pathways, but the goal of routine and comprehensive overexpression screens has been hampered by the lack of systematic libraries. Here we describe the construction of a systematic collection of the Saccharomyces cerevisiae genome in a high-copy vector and its validation in two overexpression screens.
BUR1 and BUR2 encode the catalytic and regulatory subunits of a cyclin-dependent protein kinase complex that is essential for normal growth and has a general role in transcription elongation. To gain insight into its specific role in vivo, we identified mutations that reverse the severe growth defect of bur1⌬ cells. This selection identified mutations in SET2, which encodes a histone methylase that targets lysine 36 of histone H3 and, like BUR1, has a poorly characterized role during transcription elongation. This genetic relationship indicates that SET2 activity is required for the growth defect observed in bur1⌬ strains. This SET2-dependent growth inhibition occurs via methylation of histone H3 on lysine 36, since a methylation-defective allele of SET2 or a histone H3 K36R mutation also suppressed bur1⌬. We have explored the relationship between BUR1 and SET2 at the biochemical level and find that histone H3 is monomethylated, dimethylated, and trimethylated on lysine 36 in wild-type cells, but trimethylation is significantly reduced in bur1 and bur2 mutant strains. A similar methylation pattern is observed in RNA polymerase II C-terminal domain truncation mutants and in an spt16 mutant strain. Chromatin immunoprecipitation assays reveal that the transcription-dependent increase in trimethylated K36 over open reading frames is significantly reduced in bur2⌬ strains. These results establish links between a regulatory protein kinase and histone methylation and lead to a model in which the Bur1-Bur2 complex counteracts an inhibitory effect of Set2-dependent histone methylation.
The cloning of the largest subunit of RNA polymerase II (pol II) from mouse and Saccharomyces cerevisiae in 1985 (3, 28) revealed a remarkable and highly conserved domain known as the pol II carboxy-terminal domain (CTD). This domain has intrigued researchers interested in the mechanisms regulating gene expression ever since its discovery, because of both its simplicity and its complexity. The CTD is simple in the sense that it consists entirely of repeats of the 7-amino-acid consensus sequence YSPTSPS. The mouse (28) and human (125) CTDs consist of 52 repeats, of which 21 exactly match the consensus while 20 differ at only a single position (Table 1). This same consensus sequence is conserved in other eukaryotes, with 27 repeats in budding yeast (18 exact and 5 with a single difference) (3) and 45 repeats in the more divergent Drosophila CTD (2 exact and 15 with a single difference) (4,133). In contrast to its simple repetitive composition, the functions of the CTD are quite complex, being involved in all major steps of mRNA formation, including transcription initiation and elongation, capping, splicing, and 3Ј end processing (30,42). With such critical roles in gene expression, it is not surprising that the CTD is essential for viability (4,8,86,133) and has been the subject of intense study.The CTD is not simply a passive component of the transcription and RNA processing machinery but also performs important regulatory roles. This regulatory aspect of the CTD was first suggested by the finding that the CTD is phosphorylated (13) and, more importantly, that phosphorylation of the CTD varies during the transcription cycle (54, 73). These insights stimulated searches for the CTD-specific kinase, but instead of a single kinase, several kinases that are capable of phosphorylating the CTD in vitro have been discovered. The goal of this review is to describe the present understanding of these candidate CTD kinases and their functions. The reader is referred to excellent comprehensive reviews for more detailed discussion regarding the role of the CTD during transcription (30) and as an organizing scaffold during mRNA synthesis (42); these topics will only be summarized here briefly as necessary. VARIABLE CTD REQUIREMENT DURING TRANSCRIPTIONTo understand the functions of the CTD kinases, it is first necessary to summarize the present view of the roles of the CTD itself. The CTD is essential for viability in mouse, yeast, and Drosophila (4,8,86,133), although mutants with deletions that remove approximately half of the repeats are still viable. CTD truncations reduce the levels of several transcripts tested in yeast (105), but not all pol II-dependent promoters are affected. This differential requirement for the CTD can be recapitulated in nuclear extracts (68) and thus is due to direct effects on transcription. Promoter-specific requirements for the CTD are not restricted to yeast, as they have also been observed using promoters derived from other organisms. A CTD-less form of pol II, for example, is active in nonspec...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.