Biomedical subjects

B Stillman

Publications and source records attributed to B Stillman.

At least 19 recordsLinked to original sources

PubMed1998

Formation of a preinitiation complex by S-phase cyclin CDK-dependent loading of Cdc45p onto chromatin.

Cdc45p, a protein essential for initiation of DNA replication, associates with chromatin after "start" in late G1 and during the S phase of the cell cycle. Binding of Cdc45p to chromatin depends on Clb-Cdc28 kinase activity as well as functional Cdc6p and Mcm2p, which suggests that Cdc45p associates with the prereplication complex after activation of S-phase cyclin-dependent kinases (CDKs). As indicated by the timing and the CDK dependence, binding of Cdc45p to chromatin is crucial for commitment to initiation of DNA replication. During S phase, Cdc45p physically interacts with minichromosome maintenance (MCM) proteins on chromatin; however, dissociation of Cdc45p from chromatin is slower than that of MCMs, which indicates that the proteins are released by different mechanisms.

CDC28 Protein Kinase, S cerevisiae↗

PubMed1998

Cdc6 is regulated by E2F and is essential for DNA replication in mammalian cells.

Cdc6 has a critical regulatory role in the initiation of DNA replication in yeasts, but its function in mammalian cells has not been characterized. We show here that Cdc6 is expressed selectively in proliferating but not quiescent mammalian cells, both in culture and within tissues of intact animals. During the transition from a growth-arrested to a proliferative state, transcription of mammalian Cdc6 is regulated by E2F proteins, as revealed by a functional analysis of the human Cdc6 promoter and by the ability of exogenously expressed E2F proteins to stimulate the endogenous Cdc6 gene. Immunodepletion of Cdc6 by microinjection of anti-Cdc6 antibody blocks initiation of DNA replication in a human tumor cell line. We conclude that expression of human Cdc6 is regulated in response to mitogenic signals though transcriptional control mechanisms involving E2F proteins, and that Cdc6 is required for initiation of DNA replication in mammalian cells.

Animals↗

PubMed1998

Reconstitution of recombinant human replication factor C (RFC) and identification of an RFC subcomplex possessing DNA-dependent ATPase activity.

Replication factor C (RFC) is a five-subunit protein complex required for coordinate leading and lagging strand DNA synthesis during S phase and DNA repair in eukaryotic cells. It functions to load the proliferating cell nuclear antigen (PCNA), a processivity factor for polymerases delta and epsilon, onto primed DNA templates. This process, which is ATP-dependent, is carried out by 1) recognition of the primer terminus by RFC () binding to and disruption of the PCNA trimer, and then 3) topologically linking the PCNA to the DNA. In this report, we describe the purification and properties of recombinant human RFC expressed in Sf9 cells from baculovirus expression vectors. Like native RFC derived from 293 cells, recombinant RFC was found to support SV40 DNA synthesis and polymerase delta DNA synthesis in vitro and to possess an ATPase activity that was highly stimulated by DNA and further augmented by PCNA. Assembly of RFC was observed to involve distinct subunit interactions in which both the 36- and 38-kDa subunits interacted with the 37-kDa subunit, and the 40-kDa subunit interacted with the 36-kDa subunit-37-kDa subunit subcomplex. The 140-kDa subunit was found to require interactions primarily with the 38- and 40-kDa subunits for incorporation into the complex. In addition, a stable subcomplex lacking the 140-kDa subunit, although defective for DNA replication, was found to possess DNA-dependent ATPase activity that was not responsive to the addition of PCNA.

Adenosine Triphosphatases↗

PubMed1998

Nucleosomal DNA regulates the core-histone-binding subunit of the human Hat1 acetyltransferase.

BACKGROUND: In eukaryotic cells, newly synthesized histone H4 is acetylated at lysines 5 and 12, a transient modification erased by deacetylases shortly after deposition of histones into chromosomes. Genetic studies in Saccharomyces cerevisiae revealed that acetylation of newly synthesized histones H3 and H4 is likely to be important for maintaining cell viability; the precise biochemical function of this acetylation is not known, however. The identification of enzymes mediating site-specific acetylation of H4 at Lys5 and Lys12 may help explain the function of the acetylation of newly synthesized histones. RESULTS: A cDNA encoding the catalytic subunit of the human Hat1 acetyltransferase was cloned and, using specific antibodies, the Hat1 holoenzyme was purified from human 293 cells. The human enzyme acetylates soluble but not nucleosomal H4 at Lys5 and Lys12 and acetylates histone H2A at Lys5. Unexpectedly, we found Hat1 in the nucleus of S-phase cells. Like its yeast counterpart, the human holoenzyme consists of two subunits: a catalytic subunit, Hat1, and a subunit that binds core histones, p46, which greatly stimulates the acetyltransferase activity of Hat1. Both p46 and the highly related p48 polypeptide (the small subunit of human chromatin assembly factor 1; CAF-1) bind directly to helix 1 of histone H4, a region that is not accessible when H4 is in chromatin. CONCLUSIONS: We suggest that p46 and p48 are core-histone-binding subunits that target chromatin assembly factors, chromatin remodeling factors, histone acetyltransferases and histone deacetylases to their histone substrates in a manner that is regulated by nucleosomal DNA.

Acetylation↗

PubMed1998

Cellular proteins required for adeno-associated virus DNA replication in the absence of adenovirus coinfection.

We previously reported the development of an in vitro adeno-associated virus (AAV) DNA replication system. The system required one of the p5 Rep proteins encoded by AAV (either Rep78 or Rep68) and a crude adenovirus (Ad)-infected HeLa cell cytoplasmic extract to catalyze origin of replication-dependent AAV DNA replication. However, in addition to fully permissive DNA replication, which occurs in the presence of Ad, AAV is also capable of partially permissive DNA replication in the absence of the helper virus in cells that have been treated with genotoxic agents. Limited DNA replication also occurs in the absence of Ad during the process of establishing a latent infection. In an attempt to isolate uninfected extracts that would support AAV DNA replication, we discovered that HeLa cell extracts grown to high density can occasionally display as much in vitro replication activity as Ad-infected extracts. This finding confirmed previous genetic analyses which suggested that no Ad-encoded proteins were absolutely essential for AAV DNA replication and that the uninfected extracts should be useful for studying the differences between helper-dependent and helper-independent AAV DNA replication. Using specific chemical inhibitors and monoclonal antibodies, as well as the fractionation of uninfected HeLa extracts, we identified several of the cellular enzymes involved in AAV DNA replication. They were the single-stranded DNA binding protein, replication protein A (RFA), the 3' primer binding complex, replication factor C (RFC), and proliferating cell nuclear antigen (PCNA). Consistent with the current model for AAV DNA replication, which requires only leading-strand DNA synthesis, we found no requirement for DNA polymerase alpha-primase. AAV DNA replication could be reconstituted with purified Rep78, RPA, RFC, and PCNA and a phosphocellulose chromatography fraction (IIA) that contained DNA polymerase activity. As both RFC and PCNA are known to be accessory proteins for polymerase delta and epsilon, we attempted to reconstitute AAV DNA replication by substituting either purified polymerase delta or polymerase epsilon for fraction IIA. These attempts were unsuccessful and suggested that some novel cellular protein or modification was required for AAV DNA replication that had not been previously identified. Finally, we also further characterized the in vitro DNA replication assay and demonstrated by two-dimensional (2D) gel electrophoresis that all of the intermediates commonly seen in vivo are generated in the in vitro system. The only difference was an accumulation of single-stranded DNA in vivo that was not seen in vitro. The 2D data also suggested that although both Rep78 and Rep68 can generate dimeric intermediates in vitro, Rep68 is more efficient in processing dimers to monomer duplex DNA. Regardless of the Rep that was used in vitro, we found evidence of an interaction between the elongation complex and the terminal repeats. Nicking at the terminal repeats of a replicating molecule appeared to be inhibited until after elongation was complete.

Adenoviridae↗

PubMed1998

Activation of chromosomal DNA replication in Saccharomyces cerevisiae by acidic transcriptional activation domains.

A large body of evidence from viral systems has established that transcription factors play an important and direct role in activating viral DNA replication. Among the transcriptional activation domains that can stimulate viral DNA replication are acidic domains such as those derived from herpes simplex virus VP16 and the tumor suppressor p53. Here we show that acidic activation domains can also activate a cellular origin of replication in a chromosomal context. When tethered to the yeast ARS1 (autonomously replicating sequence 1) origin of replication, both VP16 and p53 activation domains can enhance origin function. In addition, the C-terminal acidic region of the yeast transcription factor ABF1, which normally activates the ARS1 origin, is sufficient for activating ARS1 function when tethered to the origin. Mutations at residues Trp-53 and Phe-54 of a 20-residue (41 to 60) activation region of p53 abolish the activation of both replication and transcription, suggesting that the same structural determinants may be employed to activate both processes in yeast. Furthermore, using a two-dimensional gel electrophoresis method, we demonstrate that the GAL4-p53 chimeric activator can activate initiation of chromosomal replication from an origin inserted at the native ARS1 locus. These findings strongly suggest functional conservation of the mechanisms used by the acidic activation domains to activate viral DNA replication in mammalian cells and chromosomal replication in yeast.

Chromosome Mapping↗

PubMed1998

Cyclin-dependent kinase inhibitor p21 modulates the DNA primer-template recognition complex.

The p21 protein, a cyclin-dependent kinase (CDK) inhibitor, is capable of binding to both cyclin-CDK and the proliferating cell nuclear antigen (PCNA). Through its binding to PCNA, p21 can regulate the function of PCNA differentially in replication and repair. To gain an understanding of the precise mechanism by which p21 affects PCNA function, we have designed a new assay for replication factor C (RFC)-catalyzed loading of PCNA onto DNA, a method that utilizes a primer-template DNA attached to agarose beads via biotin-streptavidin. Using this assay, we showed that RFC remains transiently associated with PCNA on the DNA after the loading reaction. Addition of p21 did not inhibit RFC-dependent PCNA loading; rather, p21 formed a stable complex with PCNA on the DNA. In contrast, the formation of a p21-PCNA complex on the DNA resulted in the displacement of RFC from the DNA. The nonhydrolyzable analogs of ATP, adenosine-5'-O-(3-thiotriphosphate) (ATPgammaS) and adenyl-imidodiphosphate, each stabilized the primer recognition complex containing RFC and PCNA in the absence of p21. RFC in the ATPgammaS-activated complex was no longer displaced from the DNA by p21. We propose that p21 stimulates the dissociation of the RFC from the PCNA-DNA complex in a process that requires ATP hydrolysis and then inhibits subsequent PCNA-dependent events in DNA replication. The data suggest that the conformation of RFC in the primer recognition complex might change on hydrolysis of ATP. We also suggest that the p21-PCNA complex that remains attached to DNA might function to tether cyclin-CDK complexes to specific regions of the genome.

Adenosine Triphosphate↗

PubMed1998

The DNA replication fork in eukaryotic cells.

Replication of the two template strands at eukaryotic cell DNA replication forks is a highly coordinated process that ensures accurate and efficient genome duplication. Biochemical studies, principally of plasmid DNAs containing the Simian Virus 40 origin of DNA replication, and yeast genetic studies have uncovered the fundamental mechanisms of replication fork progression. At least two different DNA polymerases, a single-stranded DNA-binding protein, a clamp-loading complex, and a polymerase clamp combine to replicate DNA. Okazaki fragment synthesis involves a DNA polymerase-switching mechanism, and maturation occurs by the recruitment of specific nucleases, a helicase, and a ligase. The process of DNA replication is also coupled to cell-cycle progression and to DNA repair to maintain genome integrity.

Cell Cycle↗

PubMed1997

Persistent initiation of DNA replication and chromatin-bound MCM proteins during the cell cycle in cdc6 mutants.

Faithful inheritance of genetic information requires that DNA be copied only once each cell cycle. Initiation of DNA replication involves the establishment of a prereplication complex (pre-RC) and subsequent activation by CDK/cyclins, converting the pre-RC to a post-RC. The origin recognition complex (ORC), Cdc6p, and the MCM proteins are required for establishing the pre-RC. We show that all six ORC subunits remain bound to chromatin throughout the cell cycle, whereas the MCM proteins cycle on and off, corresponding precisely to transitions of the RC. A newly isolated cdc6 mutant displays promiscuous initiation of DNA replication, increased nuclear DNA content, and constant MCM protein association with chromatin throughout the cell cycle. This gain-of-function cdc6 mutant ignores the negative controls imposed normally on initiation by the CDK/cyclins, suggesting that Cdc6p is a key mediator of once-per-cell-cycle control of DNA replication.

CDC28 Protein Kinase, S cerevisiae↗

PubMed1997

Inhibition of CDK activity and PCNA-dependent DNA replication by p21 is blocked by interaction with the HPV-16 E7 oncoprotein.

p21 inhibits cyclin-dependent kinase (CDK) activity and proliferating cell nuclear antigen (PCNA)-dependent DNA replication by binding to CDK/cyclin complexes and to PCNA through distinct domains. The human papillomavirus (HPV)-16 E7 oncoprotein (16E7) abrogated a DNA damage-induced cell cycle arrest in vivo, despite high levels of p21. Using cell lysates and purified proteins we show that 16E7 prevented p21 both from inhibiting CDK2/cyclin E activity and PCNA-dependent DNA replication, whereas the nononcogenic HPV-6 E7 had reduced effects. Inactivation of both inhibitory functions of p21 was attained through binding between 16E7 and sequences in the carboxy-terminal end of p21 that overlap with the PCNA-binding site and the second p21 cyclin-binding motif. These data imply that the carboxyl terminus of p21 simultaneously modulates both CDK activity and PCNA-dependent DNA replication and that a single protein, 16E7, can override this modulation to disrupt normal cell cycle control.

Binding Sites↗

PubMed1997

Ultraviolet radiation sensitivity and reduction of telomeric silencing in Saccharomyces cerevisiae cells lacking chromatin assembly factor-I.

In vivo, nucleosomes are formed rapidly on newly synthesized DNA after polymerase passage. Previously, a protein complex from human cells, termed chromatin assembly factor-I (CAF-I), was isolated that assembles nucleosomes preferentially onto SV40 DNA templates that undergo replication in vitro. Using a similar assay, we now report the purification of CAF-I from the budding yeast Saccharomyces cerevisiae. Amino acid sequence data from purified yeast CAF-I led to identification of the genes encoding each subunit in the yeast genome data base. The CAC1 and CAC2 (chromatin assembly complex) genes encode proteins similar to the p150 and p60 subunits of human CAF-I, respectively. The gene encoding the p50 subunit of yeast CAF-I (CAC3) is similar to the human p48 CAF-I subunit and was identified previously as MSI1, a member of a highly conserved subfamily of WD repeat proteins implicated in histone function in several organisms. Thus, CAF-I has been conserved functionally and structurally from yeast to human cells. Genes encoding the CAF-I subunits (collectively referred to as CAC genes) are not essential for cell viability. However, deletion of any CAC gene causes an increase in sensitivity to ultraviolet radiation, without significantly increasing sensitivity to gamma rays. This is consistent with previous biochemical data demonstrating the ability of CAF-I to assemble nucleosomes on templates undergoing nucleotide excision repair. Deletion of CAC genes also strongly reduces silencing of genes adjacent to telomeric DNA; the CAC1 gene is identical to RLF2 (Rap1p localization factor-2), a gene required for the normal distribution of the telomere-binding Rap1p protein within the nucleus. Together, these data suggest that CAF-I plays a role in generating chromatin structures in vivo.

Amino Acid Sequence↗

PubMed1997

p62(dok): a constitutively tyrosine-phosphorylated, GAP-associated protein in chronic myelogenous leukemia progenitor cells.

Characteristic of chronic myelogenous leukemia (CML) is the presence of the chimeric p210(bcr-abl) protein possessing elevated protein tyrosine kinase activity relative to normal c-abl tyrosine kinase. Hematopoietic progenitors isolated from CML patients in the chronic phase contain a constitutively tyrosine-phosphorylated protein that migrates at 62 kDa by SDS-PAGE and associates with the p120 ras GTPase-activating protein (GAP). We have purified p62(dok) from a hematopoietic cell line expressing p210(bcr-abl). p62(dok) is a novel protein with features of a signaling molecule. Association of p62(dok) with GAP correlates with its tyrosine phosphorylation. p62(dok) is rapidly tyrosine-phosphorylated upon activation of the c-Kit receptor, implicating it as a component of a signal transduction pathway downstream of receptor tyrosine kinases.

CSK Tyrosine-Protein Kinase↗

PubMed1997

A human protein related to yeast Cdc6p.

The unstable proteins Cdc6p and cdc18+ are essential and rate limiting for the initiation of DNA replication in Saccharomyces cerevisiae and Schizosaccharomyces pombe, respectively, and also participate in checkpoint controls that ensure DNA replication is completed before mitosis is initiated. We have identified Xenopus and human proteins closely related to Cdc6p/cdc18. The human protein, p62(cdc6), is encoded on chromosome 17q21.3 and includes putative cyclin-dependent kinase phosphorylation sites, destruction boxes, a nucleotide binding/ATPase domain, and a potential leucine zipper. Expression of p62(cdc6) mRNA and protein is suppressed in human diploid fibroblasts made quiescent by serum starvation, and peaks as cells reenter the cell cycle and replicate DNA following serum stimulation. Conservation of structure among proteins involved in initiation suggests that fundamental features of replication complexes are maintained in all eukaryotes.

Amino Acid Sequence↗

PubMed1997

CDC45, a novel yeast gene that functions with the origin recognition complex and Mcm proteins in initiation of DNA replication.

The CDC45 gene of Saccharomyces cerevisiae was isolated by complementation of the cold-sensitive cdc45-1 mutant and shown to be essential for cell viability. Although CDC45 genetically interacts with a group of MCM genes (CDC46, CDC47, and CDC54), the predicted sequence of its protein product reveals no significant sequence similarity to any known Mcm family member. Further genetic characterization of the cdc45-1 mutant demonstrated that it is synthetically lethal with orc2-1, mcm2-1, and mcm3-1. These results not only reveal a functional connection between the origin recognition complex (ORC) and Cdc45p but also extend the CDC45-MCM genetic interaction to all known MCM family members that were shown to be involved in replication initiation. Initiation of DNA replication in cdc45-1 cells was defective, causing a delayed entry into S phase at the nonpermissive temperature, as well as a high plasmid loss rate which could be suppressed by tandem copies of replication origins. Furthermore, two-dimensional gels directly showed that chromosomal origins fired less frequently in cdc45-1 cells at the nonpermissive temperature. These findings suggest that Cdc45p, ORC, and Mcm proteins act in concert for replication initiation throughout the genome.

Amino Acid Sequence↗

PubMed1997

p53-induced p21 controls DNA replication.

The p21 protein, a regulator of cyclin-dependent kinases (CDKs), has been thought to be one of the key proteins to function in cell proliferation suppression upon DNA damage. In normal cells but not in many tumor cells, p21 forms a quaternary complex with a cyclin, a CDK and the proliferating cell nuclear antigen (PCNA), one of the DNA replication and repair factors, suggesting that this complex might play an important role in maintaining the integrity of the genome. Here, we have focused on the p21-PCNA interaction in the context of DNA replication or DNA repair, presenting the data from both in vitro and in vivo studies of the p21 function.

Antigens, Viral, Tumor↗

PubMed1996

Cell cycle control of DNA replication.

The initiation of DNA replication in eukaryotic cells is a highly regulated process that leads to the duplication of the genetic information for the next cell generation. This requires the ordered assembly of many proteins at the origins of DNA replication to form a competent, pre-replicative chromosomal state. In addition to this competent complex, at least two cell cycle regulated protein kinase pathways are required to affect a transition to a post-replicative chromosomal state. Protein kinases required to establish mitosis prevent re-replication of the DNA. As cells exit mitosis, the cell cycle is reset, allowing the establishment of a new, competent replication state.

Animals↗

PubMed1996

Nucleosome assembly by a complex of CAF-1 and acetylated histones H3/H4.

Chromatin assembly factor 1 (CAF-1) assembles nucleosomes in a replication-dependent manner. The small subunit of CAF-1 (p48) is a member of a highly conserved subfamily of WD-repeat proteins. There are at least two members of this subfamily in both human (p46 and p48) and yeast cells (Hat2p, a subunit of the B-type H4 acetyltransferase, and Msi1p). Human p48 can bind to histone H4 in the absence of CAF-1 p150 and p60. p48, also a known subunit of a histone deacetylase, copurifies with a chromatin assembly complex (CAC), which contains the three subunits of CAF-1 (p150, p60, p48) and H3 and H4, and promotes DNA replication-dependent chromatin assembly. CAC histone H4 exhibits a novel pattern of lysine acetylation that overlaps with, but is distinct from, that reported for newly synthesized H4 isolated from nascent chromatin. Our data suggest that CAC is a key intermediate of the de novo nucleosome assembly pathway and that the p48 subunit participates in other aspects of histone metabolism.

Acetylation↗

PubMed1996

Chromatin assembly coupled to DNA repair: a new role for chromatin assembly factor I.

DNA repair in the eukaryotic cell disrupts local chromatin organization. To investigate whether the resetting of nucleosomal arrays can be linked to the repair process, we developed model systems, with both Xenopus egg extract and human cell extracts, to follow repair and chromatin assembly in parallel on circular DNA templates. Both systems were able to carry out nucleotide excision repair of DNA lesions. We observed that UV-dependent DNA synthesis occurs simultaneously with chromatin assembly, strongly indicating a mechanistic coupling between the two processes. A complementation assay established that chromatin assembly factor I (CAF1) is necessary for this repair associated chromatin formation.

Animals↗