The Saccharomyces cerevisiae DNA Polymerase α Catalytic Subunit Interacts with Cdc68/Spt16 and with Pob3, a Protein Similar to an HMG1-Like Protein

Abstract

We have used DNA polymerase ␣ affinity chromatography to identify factors involved in eukaryotic DNA replication in the yeast Saccharomyces cerevisiae. Two proteins that bound to the catalytic subunit of DNA polymerase ␣ (Pol1 protein) are encoded by the essential genes CDC68/SPT16 and POB3. The binding of both proteins was enhanced when extracts lacking a previously characterized polymerase binding protein, Ctf4, were used. This finding suggests that Cdc68 and Pob3 may compete with Ctf4 for binding to Pol1. Pol1 and Pob3 were coimmunoprecipitated from whole-cell extracts with antiserum directed against Cdc68, and Pol1 was immunoprecipitated from whole-cell extracts with antiserum directed against the amino terminus of Pob3, suggesting that these proteins may form a complex in vivo. CDC68 also interacted genetically with POL1 and CTF4 mutations; the maximum permissive temperature of double mutants was lower than for any single mutant. Overexpression of Cdc68 in a pol1 mutant strain dramatically decreased cell viability, consistent with the formation or modulation of an essential complex by these proteins in vivo. A mutation in CDC68/SPT16 had previously been shown to cause pleiotropic effects on the regulation of transcription (J. The cdc68-1 mutation caused elevated rates of chromosome fragment loss, a phenotype characteristic of genes whose native products are required for normal DNA metabolism. However, this mutation did not affect the rate of loss or recombination for two intact chromosomes, nor did it affect the retention of a low-copy-number plasmid. The previously uncharacterized Pob3 sequence has significant amino acid sequence similarity with an HMG1-like protein from vertebrates. Based on these results and because Cdc68 has been implicated as a regulator of chromatin structure, we postulate that polymerase ␣ may interact with these proteins to gain access to its template or to origins of replication in vivo.ADNA replication is accomplished by large multiprotein complexes called replisomes, as demonstrated in studies using the replication machinery from Escherichia coli and its bacteriophages (39). The E. coli polymerase III holoenzyme is unusual in that it is stable enough to be purified intact, allowing its components to be identified (39). However, in most other systems the replisome is unstable, and components have been identified by using partial-function assays, genetic methods, in vitro complementation of extracts prepared from replicationdefective mutants, or affinity chromatography (39, 73). Studies of the replication machinery used by the eukaryotic virus simian virus 40 (SV40) showed that functional units in eukaryotic replisomes are remarkably similar to those found in prokaryotic systems (73). Although the proteins typically have little similarity at the amino acid level, the need for polymerases, processivity clamps, clamp loading proteins, helicases, primases, nucleases, and single-stranded DNA binding proteins appears to be universal (29,39,73). Some physical interactions have been detec...

The Histone Chaperone Facilitates Chromatin Transcription (FACT) Protein Maintains Normal Replication Fork Rates

“…As described under "Functional Roles of FACT in DNA Replication Initiation Versus Elongation," FACT interacts with Pol␣, RPA, and the MCM complex (28,31,32) and apparently facilitates replication fork progression via its functional interaction with these replication enzymes/factors. This possibility seems to be supported by a previous study (32) that demonstrated that a point mutation introduced within a conserved surface cluster in the budding yeast Pob3-M, corresponding to the part of the Pob3 domain without the acidic region of the SSRP1 subunit of FACT (Figs.…”

Multiple Nhp6 Molecules Are Required to Recruit Spt16-Pob3 to Form yFACT Complexes and to Reorganize Nucleosomes

“…Nhp6 supports the function of Spt16-Pob3, a heterodimer implicated in both DNA replication and regulation of transcription (10 -12). Spt16 and Pob3 contain most of the sequences found in the components of the human FACT and frog DUF complexes (13,14), but Pob3 lacks the HMG box motif found in the otherwise homologous FACT subunit SSRP1 (12,15,16). Nhp6 appears to provide HMG box function for Spt16-Pob3 both in vivo and in vitro as cells lacking Nhp6 cannot tolerate mutations in SPT16 or POB3 (11,17), and Spt16-Pob3 can only bind to and reorganize nucleosomes in vitro if Nhp6 is present (11).…”

Coupling caspase cleavage and ubiquitin–proteasome-dependent degradation of SSRP1 during apoptosis

“…31 In yeast, Pob3, the N-terminal counterpart of SSRP1, is also strictly required, as Pob3-null strains are not viable. 9 More precisely, as a subunit of FACT, SSRP1 appears to be critical for transcription in a wide variety of organisms, from yeast to mammals. 7 FACT is also involved in replication in yeast 9 and xenopus.…”

“…9 More precisely, as a subunit of FACT, SSRP1 appears to be critical for transcription in a wide variety of organisms, from yeast to mammals. 7 FACT is also involved in replication in yeast 9 and xenopus. 11 Furthermore, SSRP1 may function in more specific pathways as a coactivator for SRF and p63.…”

“…8 In yeast and xenopus, FACT is directly involved in DNA replication. [9][10][11] Fewer studies have explored the role of FACT in recombination 12 and DNA-damage response. Our lab recently showed that ultraviolet (UV) irradiation causes human FACT to associate with casein kinase 2, which results in the DNA-damage-dependent phosphorylation and activation of p53.…”