Biomedical subjects
Publications and source records attributed to Z Kelman.
Replicative DNA polymerases are highly processive enzymes that polymerize thousands of nucleotides without dissociating from the DNA template. The recently determined structure of the Escherichia coli bacteriophage T7 DNA polymerase suggests a unique mechanism that underlies processivity, and this mechanism may generalize to other replicative polymerases.
Proliferating-cell nuclear antigen (PCNA) plays an essential role in nucleic-acid metabolism in all eukaryotes. The PCNA protein interacts with a large number of proteins. These proteins can be divided into two groups: the first contains proteins that have a known enzymatic activity; the second contains regulatory proteins that are involved in cell-cycle progression, checkpoint control and cellular differentiation. Interestingly, all of the enzymes known to interact with PCNA either recognize specific structures on DNA or have limited DNA-sequence specificity. Proteins that have low sequence specificities could utilize PCNA as an adapter in order to interact with their DNA substrates.
DNA polymerase delta (pol delta) plays an essential role in DNA replication, repair, and recombination. We have purified pol delta from Schizosaccharomyces pombe more than 10(3)-fold and demonstrated that the polymerase activity of purified S. pombe pol delta is completely dependent on proliferating cell nuclear antigen and replication factor C. SDS/PAGE analysis of the purified fraction indicated that the pol delta complex consists of five subunits that migrate with apparent molecular masses of 125, 55, 54, 42, and 22 kDa. Western blot analysis indicated that the 125, 55, and 54 kDa proteins are the large catalytic subunit (Pol3), Cdc1, and Cdc27, respectively. The identity of the other two subunits, p42 and p22, was determined following proteolytic digestion and sequence analysis of the resulting peptides. The peptide sequences derived from the p22 subunit indicated that this subunit is identical to Cdm1, previously identified as a multicopy suppressor of the temperature-sensitive cdc1-P13 mutant, whereas peptide sequences derived from the p42 subunit were identical to a previously uncharacterized ORF located on S. pombe chromosome 1.
Proliferating cell nuclear antigen (PCNA) plays an essential role in nucleic acid metabolism as a component of the replication and repair machinery. This toroidal-shaped protein encircles DNA and can slide bidirectionally along the duplex. One of the well-established functions for PCNA is its role as the processivity factor for DNA polymerase delta and epsilon. PCNA tethers the polymerase catalytic unit to the DNA template for rapid and processive DNA synthesis. In the last several years it has become apparent that PCNA interacts with proteins involved in cell-cycle progression which are not a part of the DNA polymerase apparatus. Some of these interactions have a direct effect on DNA synthesis while the roles of several other interactions are not fully understood. This review summarizes the structural features of PCNA and describes the diverse functions played by the protein in DNA replication and repair as well as its possible role in chromatin assembly and gene transcription. The PCNA interactions with different cellular proteins and the importance of these interactions are also discussed.
In eukaryotes, processive DNA synthesis catalyzed by DNA polymerases delta and epsilon (pol delta and epsilon) requires the proliferating cell nuclear antigen (PCNA). It has recently been shown that in humans (h), the PCNA function, required for both DNA replication and nucleotide excision repair, can be inactivated by p21(CIP1) due to a specific interaction between hPCNA and the carboxyl terminus of p21(CIP1). In this report, we show that Saccharomyces cerevisiae (S. cerevisiae) PCNA-dependent pol delta-catalyzed DNA synthesis was inhibited less efficiently than the human system by the intact p21(CIP1) protein and was unaffected by the p21(CIP1) carboxyl-terminal peptide (codons 139-160). This species-specific response of PCNA to p21(CIP1)-mediated inhibition of DNA synthesis results from a marked difference in the ability of h and S. cerevisiae PCNA to interact with p21(CIP1). As shown by binding studies using the surface plasmon resonance technique, hPCNA binds both full-length p21(CIP1) and the p21(CIP1) peptide-(139-160) stoichiometrically with a similar affinity (KD approximately 2.5 nM) while S. cerevisiae PCNA binds p21(CIP1) with approximately 10-fold less affinity and does not interact with the p21(CIP1) peptide-(139-160).
A "minimal" DNA primer-template system, consisting of an 80-mer template and 30-mer primer, supports processive DNA synthesis by DNA polymerase III core in the presence of the beta sliding clamp, gamma complex clamp loader, and single-stranded binding protein from Escherichia coli. This primer-template system was used to measure the loading of the beta sliding clamp by the gamma complex in an ATP-dependent reaction. Bound protein-DNA complexes were detected by monitoring fluorescence depolarization of DNA. Steady state and time-resolved anisotropies were measured, and stopped-flow pre-steady state fluorescence measurements allowed visualization of the loading reactions in real time. The rate of loading beta onto DNA was 12 s-1, demonstrating that clamp assembly is rapid on the time scale required for lagging strand Okazaki fragment synthesis. The association rate appears to be limited by an intramolecular step occurring prior to the clamp-loading reaction, possibly the opening of the toroidal beta dimer.
The crystal structure of the human DNA polymerase delta processivity factor PCNA (proliferating cell nuclear antigen) complexed with a 22 residue peptide derived from the C-terminus of the cell-cycle checkpoint protein p21(WAF1/CIP1) has been determined at 2.6 angstrom resolution. p21 binds to PCNA in a 1:1 stoichiometry with an extensive array of interactions that include the formation of a beta sheet with the interdomain connector loop of PCNA. An intact trimeric ring is maintained in the structure of the p21-PCNA complex, with a central hole available for DNA interaction. The ability of p21 to inhibit the action of PCNA is therefore likely to be due to its masking of elements on PCNA that are required for the binding of other components of the polymerase assembly.
BACKGROUND: The high speed and processivity of replicative DNA polymerases reside in a processivity factor which has been shown to be a ring-shaped protein. This protein ("sliding clamp') encircles DNA and tethers the catalytic unit to the template. Although in eukaryotic, prokaryotic and bacteriophage-T4 systems, the processivity factors are ring-shaped, they assume different oligomeric states. The Escherichia coli clamp (the beta subunit) is active as a dimer while the eukaryotic and T4 phage clamps (PCNA and gp45, respectively) are active as trimers. The clamp can not assemble itself on DNA. Instead, a protein complex known as a clamp loader utilizes ATP to assemble the ring around the primer-template. This study compares properties of the human PCNA clamp with those of E. coli and T4 phage. RESULTS: The PCNA ring is a stable trimer down to a concentration below 100 nM (Kd approximately 21 nM). On DNA, the PCNA clamp slides freely and dissociates from DNA slowly (t1/2 approximately 24 min). beta is more stable in solution (Kd < 60 PM) and on DNA (t1/2 approximately 1 h) than PCNA which may be explained by its simpler oligomeric state. The T4 gp45 clamp is a much less stable trimer than PCNA (Kd approximately 250 nM) and requires association with the polymerase to stabilize it on DNA as observed previously. The consequence of this cooperation between clamp and polymerase is that upon finishing a template and dissociation of the polymerase from DNA, the gp45 clamp spontaneously dissociates from DNA without assistance. However, the greater stability of the PCNA and beta clamps on DNA necessitates an active process for their removal. The clamp loaders (RFC and gamma complex) were also capable of unloading their respective clamps from DNA in the presence of ATP. CONCLUSIONS: The stability of the different clamps in solution correlates with their stability on DNA. Thus, the low stability of the T4 clamp explains the inability to isolate gp45 on DNA. The stability of the PCNA and beta clamps predicts they will require an unloading factor to recycle them on and off DNA during replication. The clamp loaders of PCNA and beta double as clamp unloaders presumably for the purpose of clamp recycling.
Escherichia coli expression vectors, based on the pET system, were constructed to allow fusion of a protein kinase (PK) recognition motif, a hemagglutinin (HG) epitope-tag and a His6-tag at the N-terminal portion of a protein of interest. The fusion proteins, that result from expression using these vectors, can be phosphorylated in vitro using cAMP-dependent PK, immunoprecipitated using monoclonal antibody against the HG-epitope, and can be rapidly purified using a Ni2+ column.
Why do proteins proteins that encircle DNA have six-fold symmetry? One important factor may be the economy in protein mass with which DNA can be encircled by six globular subunits arranged in a ring.
The remarkable processivity of cellular replicative DNA polymerases derive their tight grip to DNA from a ring-shaped protein that encircles DNA and tethers the polymerase to the chromosome. The crystal structures of prototypical 'sliding clamps' of prokaryotes (beta subunit) and eukaryotes (PCNA) are ring shaped proteins for encircling DNA. Although beta is a dimer and PCNA is a trimer, their structures are nearly superimposable. Even though they are not hexamers, the sliding clamps have a pseudo 6-fold symmetry resulting from three globular domains comprising each beta monomer and two domains comprising each PCNA monomer. These domains have the same chain fold and are nearly identical in three-dimensions. The amino acid sequences of 11 beta and 13 PCNA proteins from different organisms have been aligned and studied to gain further insight into the relation between the structure and function of these sliding clamps. Furthermore, a putative embryonic form of PCNA is the size of beta and thus may encircle DNA as a dimer like the prokaryotic clamps.
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DNA polymerase III holoenzyme contains two DNA polymerases embedded in a particle with 9 other subunits. This multisubunit DNA polymerase is the Eschericia coli chromosomal replicase, and it has several special features that distinguish it as a replicating machine. For example, one of its subunits is a circular protein that slides along DNA while clamping the rest of the machinery to the template. Other subunits act together as a matchmaker to assemble the ring onto DNA. Overall, E. coli DNA polymerase III holoenzyme is very similar in both structure and function to the chromosomal replicases of eukaryotes, from yeast all the way up to humans. This review summarizes our present knowledge about the function of the 10 subunits of this replicating machine and how they coordinate their actions for smooth duplication of chromosomes.
Cdk-interacting protein 1 (Cip1) is a p53-regulated 21-kDa protein that inhibits several members of the cyclin-dependent kinase (CDK) family. It was initially observed in complexes containing CDK4, cyclin D, and proliferating cell nuclear antigen (PCNA). PCNA, in conjunction with activator 1, acts as a processivity factor for eukaryotic DNA polymerase (pol) delta, and these three proteins constitute the pol delta holoenzyme. In this report, we demonstrate that Cip1 can also directly inhibit DNA synthesis in vitro by binding to PCNA. Cip1 efficiently inhibits simian virus 40 replication dependent upon pol alpha, activator 1, PCNA, and pol delta, and this inhibition can be overcome by additional PCNA. Simian virus 40 DNA replication, catalyzed solely by high levels of pol alpha-primase complex, is unaffected by Cip1. Using the surface plasmon resonance technique, a direct physical interaction of PCNA and Cip1 was detected. We have observed that Cip1 efficiently inhibits synthesis of long (7.2 kb) but not short (10 nt) templates, suggesting that its association with PCNA is likely to impair the processive movement of pol delta during DNA chain elongation, as opposed to blocking assembly of the pol delta holoenzyme. The implications of the Cip1-PCNA interaction with respect to regulation of DNA synthesis, cell cycle checkpoint control, and DNA repair are discussed.
Research into the enzymology of DNA replication has seen a multitude of highly significant advances during the past year, in both prokaryotic and eukaryotic systems. The scope of this article is limited to chromosomal replicases and origins of initiation. The multiprotein chromosomal replicases of prokaryotes and eukaryotes appear to be strikingly similar in structure and function, although future work may reveal their differences. Recent developments, elaborating the activation of origins in several systems, have begun to uncover mechanisms of regulation. The enzymology of eukaryotic origins has, until now, been limited to viral systems, but over the past few years, enzymology has caught a grip on the cellular origins of yeast.
Receptor tyrosine kinases are involved in the regulation of cell growth and may play a central role in embryonic development. We recently developed a polymerase chain reaction (PCR)-based gene cloning procedure that allows selective isolation of genes that encode novel transmembrane tyrosine kinases in tissues of embryonic origin. By employing this protocol on mRNA from a 12.5 day post-coitum mouse placenta, we identified a gene for a putative receptor protein kinase. The deduced amino acid sequence predicts the existence of an approximately 200 amino acid long extracellular domain that shows no similarity to known proteins. The cytoplasmic portion contains a core sequence that is structurally homologous to the tyrosine kinase family. However, a few highly conserved short blocks of sequences, shared by all protein kinases, display variations in the isolated gene. These include the glycine-rich block at the nucleotide-binding cleft and the Asp-Phe-Gly triplet at the substrate recognition site. On the basis of these variations, we named the gene vik for variant in the kinase. Northern analysis revealed two widely expressed transcripts of vik with molecular weights of 3 and 2.5 kb. Chromosomal mapping using restriction fragment length polymorphism localized the gene to murine chromosome 9. The unique structural landmarks of vik at both the extracellular and the cytoplasmic domains suggest novel ligand as well as substrate specificity of the presumed receptor.