scholarly journals Mutations in yeast proliferating cell nuclear antigen define distinct sites for interaction with DNA polymerase delta and DNA polymerase epsilon.

1997 ◽  
Vol 17 (11) ◽  
pp. 6367-6378 ◽  
Author(s):  
J C Eissenberg ◽  
R Ayyagari ◽  
X V Gomes ◽  
P M Burgers
Keyword(s):  
Dna Synthesis ◽  
Gene Product ◽  
Dna Polymerase ◽  
Proliferating Cell Nuclear Antigen ◽  
Site Directed Mutagenesis ◽  
Nuclear Antigen ◽  
Stem Loop ◽  
Factor C ◽  
Proliferating Cell

The importance of the interdomain connector loop and of the carboxy-terminal domain of Saccharomyces cerevisiae proliferating cell nuclear antigen (PCNA) for functional interaction with DNA polymerases delta (Poldelta) and epsilon (Pol epsilon) was investigated by site-directed mutagenesis. Two alleles, pol30-79 (IL126,128AA) in the interdomain connector loop and pol30-90 (PK252,253AA) near the carboxy terminus, caused growth defects and elevated sensitivity to DNA-damaging agents. These two mutants also had elevated rates of spontaneous mutations. The mutator phenotype of pol30-90 was due to partially defective mismatch repair in the mutant. In vitro, the mutant PCNAs showed defects in DNA synthesis. Interestingly, the pol30-79 mutant PCNA (pcna-79) was most defective in replication with Poldelta, whereas pcna-90 was defective in replication with Pol epsilon. Protein-protein interaction studies showed that pcna-79 and pcna-90 failed to interact with Pol delta and Pol epsilon, respectively. In addition, pcna-90 was defective in interaction with the FEN-1 endo-exonuclease (RTH1 product). A loss of interaction between pcna-79 and the smallest subunit of Poldelta, the POL32 gene product, implicates this interaction in the observed defect with the polymerase. Neither PCNA mutant showed a defect in the interaction with replication factor C or in loading by this complex. Processivity of DNA synthesis by the mutant holoenzyme containing pcna-79 was unaffected on poly(dA) x oligo(dT) but was dramatically reduced on a natural template with secondary structure. A stem-loop structure with a 20-bp stem formed a virtually complete block for the holoenzyme containing pcna-79 but posed only a minor pause site for wild-type holoenzyme, indicating a function of the POL32 gene product in allowing replication past structural blocks.

Involvement of proliferating cell nuclear antigen (cyclin) in DNA replication in living cells

1989 ◽  
Vol 9 (1) ◽  
pp. 57-66
Author(s):  
M Zuber ◽  
E M Tan ◽  
M Ryoji
Keyword(s):  
Dna Polymerase ◽  
Proliferating Cell Nuclear Antigen ◽  
Living Cells ◽  
Nuclear Antigen ◽  
Plasmid Replication ◽  
Dna Polymerase Delta ◽  
Dna Polymerase Alpha ◽  
Proliferating Cell

Proliferating cell nuclear antigen (PCNA) (also called cyclin) is known to stimulate the activity of DNA polymerase delta but not the other DNA polymerases in vitro. We injected a human autoimmune antibody against PCNA into unfertilized eggs of Xenopus laevis and examined the effects of this antibody on the replication of injected plasmid DNA as well as egg chromosomes. The anti-PCNA antibody inhibited plasmid replication by up to 67%, demonstrating that PCNA is involved in plasmid replication in living cells. This result further implies that DNA polymerase delta is necessary for plasmid replication in vivo. Anti-PCNA antibody alone did not block plasmid replication completely, but the residual replication was abolished by coinjection of a monoclonal antibody against DNA polymerase alpha. Anti-DNA polymerase alpha alone inhibited plasmid replication by 63%. Thus, DNA polymerase alpha is also required for plasmid replication in this system. In similar studies on the replication of egg chromosomes, the inhibition by anti-PCNA antibody was only 30%, while anti-DNA polymerase alpha antibody blocked 73% of replication. We concluded that the replication machineries of chromosomes and plasmid differ in their relative content of DNA polymerase delta. In addition, we obtained evidence through the use of phenylbutyl deoxyguanosine, an inhibitor of DNA polymerase alpha, that the structure of DNA polymerase alpha holoenzyme for chromosome replication is significantly different from that for plasmid replication.

Dual mode of interaction of DNA polymerase ϵ with proliferating cell nuclear antigen in primer binding and DNA synthesis 1 1Edited by J. Karn

1999 ◽  
Vol 285 (1) ◽  
pp. 259-267 ◽  
Author(s):  
Giovanni Maga ◽  
Zophonı́as O Jónsson ◽  
Manuel Stucki ◽  
Silvio Spadari ◽  
Ulrich Hübscher
Keyword(s):  
Dna Synthesis ◽  
Dna Polymerase ◽  
Proliferating Cell Nuclear Antigen ◽  
Nuclear Antigen ◽  
Dual Mode ◽  
Proliferating Cell

scholarly journals DNA Polymerases BI and D from the Hyperthermophilic Archaeon Pyrococcus furiosus Both Bind to Proliferating Cell Nuclear Antigen with Their C-Terminal PIP-Box Motifs

2007 ◽  
Vol 189 (15) ◽  
pp. 5652-5657 ◽  
Author(s):  
Kazuo Tori ◽  
Megumi Kimizu ◽  
Sonoko Ishino ◽  
Yoshizumi Ishino
Keyword(s):  
Dna Synthesis ◽  
Proliferating Cell Nuclear Antigen ◽  
Pyrococcus Furiosus ◽  
Dna Polymerases ◽  
Nuclear Antigen ◽  
Hyperthermophilic Archaeon ◽  
Sliding Clamp ◽  
Replication Fork Progression ◽  
Proliferating Cell

ABSTRACT Proliferating cell nuclear antigen (PCNA) is the sliding clamp that is essential for the high processivity of DNA synthesis during DNA replication. Pyrococcus furiosus, a hyperthermophilic archaeon, has at least two DNA polymerases, polymerase BI (PolBI) and PolD. Both of the two DNA polymerases interact with the archaeal P. furiosus PCNA (PfuPCNA) and perform processive DNA synthesis in vitro. This phenomenon, in addition to the fact that both enzymes display 3′-5′ exonuclease activity, suggests that both DNA polymerases work in replication fork progression. We demonstrated here that both PolBI and PolD functionally interact with PfuPCNA at their C-terminal PIP boxes. The mutant PolBI and PolD enzymes lacking the PIP-box sequence do not respond to the PfuPCNA at all in an in vitro primer extension reaction. This is the first experimental evidence that the PIP-box motif, located at the C termini of the archaeal DNA polymerases, is actually critical for PCNA binding to form a processive DNA-synthesizing complex.

scholarly journals Purification and characterization of a 100 kDa DNA polymerase from cauliflower inflorescence

1998 ◽  
Vol 332 (2) ◽  
pp. 557-563 ◽  
Author(s):  
Hirokazu SETO ◽  
Masami HATANAKA ◽  
Seisuke KIMURA ◽  
Masahiko OSHIGE ◽  
Yuri TSUYA ◽  
...  
Keyword(s):  
Dna Synthesis ◽  
Dna Polymerase ◽  
Proliferating Cell Nuclear Antigen ◽  
Nuclear Antigen ◽  
Purification And Characterization ◽  
Synthetic Dna ◽  
Different Types ◽  
Proliferating Cell

A DNA polymerase from cauliflower (Brassica oleracea var. botrytis) inflorescence has been purified to near homogeneity through five successive column chromatographies, and temporally designated cauliflower polymerase 1. Cauliflower polymerase 1 is a monopolypeptide with a molecular mass of 100 kDa. The enzyme efficiently uses synthetic DNA homopolymers and moderately activated DNA and a synthetic RNA homopolymer as template-primers. The enzyme is strongly sensitive to dideoxythymidine triphosphate and N-ethylmaleimide, but it is insensitive to aphidicolin. It was stimulated with 250 mM KCl. Its mode of DNA synthesis is high-processive with or without proliferating-cell nuclear antigen. A 3´ → 5´ exonuclease activity is associated with cauliflower polymerase 1. The enzyme is clearly different from cauliflower mitochondrial polymerase and does not resemble the four different types of wheat DNA polymerase, designated wheat DNA polymerases A, B, CI and CII. In the present paper the role of the enzyme in plant DNA synthesis is discussed.

scholarly journals Functional Interactions of a Homolog of Proliferating Cell Nuclear Antigen with DNA Polymerases inArchaea

1999 ◽  
Vol 181 (21) ◽  
pp. 6591-6599 ◽  
Author(s):  
Isaac K. O. Cann ◽  
Sonoko Ishino ◽  
Ikuko Hayashi ◽  
Kayoko Komori ◽  
Hiroyuki Toh ◽  
...  
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Proliferating Cell Nuclear Antigen ◽  
Dna Polymerases ◽  
Nuclear Antigen ◽  
Pol Ii ◽  
Circular Dna ◽  
Pol I ◽  
Factor C ◽  
Proliferating Cell

ABSTRACT Proliferating cell nuclear antigen (PCNA) is an essential component of the DNA replication and repair machinery in the domainEucarya. We cloned the gene encoding a PCNA homolog (PfuPCNA) from an euryarchaeote, Pyrococcus furiosus, expressed it in Escherichia coli, and characterized the biochemical properties of the gene product. The protein PfuPCNA stimulated the in vitro primer extension abilities of polymerase (Pol) I and Pol II, which are the two DNA polymerases identified in this organism to date. An immunological experiment showed that PfuPCNA interacts with both Pol I and Pol II. Pol I is a single polypeptide with a sequence similar to that of family B (α-like) DNA polymerases, while Pol II is a heterodimer. PfuPCNA interacted with DP2, the catalytic subunit of the heterodimeric complex. These results strongly support the idea that the PCNA homolog works as a sliding clamp of DNA polymerases in P. furiosus, and the basic mechanism for the processive DNA synthesis is conserved in the domainsBacteria, Eucarya, and Archaea. The stimulatory effect of PfuPCNA on the DNA synthesis was observed by using a circular DNA template without the clamp loader (replication factor C [RFC]) in both Pol I and Pol II reactions in contrast to the case of eukaryotic organisms, which are known to require the RFC to open the ring structure of PCNA prior to loading onto a circular DNA. Because RFC homologs have been found in the archaeal genomes, they may permit more efficient stimulation of DNA synthesis by archaeal DNA polymerases in the presence of PCNA. This is the first stage in elucidating the archaeal DNA replication mechanism.

scholarly journals Mutant DNA polymerase δ from thermosensitive Schizosaccharomyces pombe strains display reduced stimulation by proliferating cell nuclear antigen

1998 ◽  
Vol 335 (3) ◽  
pp. 581-588 ◽  
Author(s):  
Mylène PERDERISET ◽  
Giovanni MAGA ◽  
Karine PIARD ◽  
Stefania FRANCESCONI ◽  
Isabelle TRATNER ◽  
...  
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Dna Polymerase ◽  
Proliferating Cell Nuclear Antigen ◽  
Catalytic Subunit ◽  
Nuclear Antigen ◽  
Wild Type ◽  
Low Levels ◽  
Proliferating Cell ◽  
Stimulation Of

We have isolated and characterized DNA polymerase δ (pol δ) from two thermosensitive Schizosaccharomyces pombe strains, polδts1 and polδts3, mutated in two different evolutionarily conserved domains of the catalytic subunit. At the restrictive temperature of 37 °C polδts1 and polδts3 mutant strains arrest growth in the S phase of the cell cycle. We show that at low levels of primer ends, in vitro stimulation by proliferating cell nuclear antigen (PCNA) of mutant enzymes is lower than stimulation of wild-type pol δ. Affinity for primer (3´-OH) ends and processivity of mutant enzymes do not appear different from wild-type pol δ. In contrast, Vmax values are lower than the wild-type value. The major in vitro defect appears to be decreased stimulation of mutant enzymes by PCNA, resulting in reduced velocity of DNA synthesis. In addition, ts1 pol δ is not stimulated by low PCNA concentration at 37 °C, although low concentrations stimulate activity at 25 °C, suggesting that this thermolability at low levels of primer ends could be its critical defect in vivo. Thus, both ts1 and ts3 pol δ mutations are located in regions of the catalytic subunit that seem necessary, directly or indirectly, for its efficient interaction with PCNA.

scholarly journals Stimulation of 3′→5′ Exonuclease and 3′-Phosphodiesterase Activities of Yeast Apn2 by Proliferating Cell Nuclear Antigen

2002 ◽  
Vol 22 (18) ◽  
pp. 6480-6486 ◽  
Author(s):  
Ildiko Unk ◽  
Lajos Haracska ◽  
Xavier V. Gomes ◽  
Peter M. J. Burgers ◽  
Louise Prakash ◽  
...  
Keyword(s):  
Proliferating Cell Nuclear Antigen ◽  
Gel Filtration ◽  
Nuclear Antigen ◽  
Binding Motif ◽  
Terminal Domain ◽  
Abasic Sites ◽  
Factor C ◽  
Proliferating Cell

ABSTRACT The Apn2 protein of Saccharomyces cerevisiae contains 3′→5′ exonuclease and 3′-phosphodiesterase activities, and these activities function in the repair of DNA strand breaks that have 3′-damaged termini and which are formed in DNA by the action of oxygen-free radicals. Apn2 also has an AP endonuclease activity and functions in the removal of abasic sites from DNA. Here, we provide evidence for the physical and functional interaction of Apn2 with proliferating cell nuclear antigen (PCNA). As indicated by gel filtration and two-hybrid studies, Apn2 interacts with PCNA both in vitro and in vivo and mutations in the consensus PCNA-binding motif of Apn2 abolish this interaction. Importantly, PCNA stimulates the 3′→5′ exonuclease and 3′-phosphodiesterase activities of Apn2. We have examined the involvement of the interdomain connector loop (IDCL) and of the carboxy-terminal domain of PCNA in Apn2 binding and found that Apn2 binds PCNA via distinct domains dependent upon whether the binding is in the absence or presence of DNA. In the absence of DNA, Apn2 binds PCNA through its IDCL domain, whereas in the presence of DNA, when PCNA has been loaded onto the template-primer junction by replication factor C, the C-terminal domain of PCNA mediates the binding.

Sign in / Sign up

Export Citation Format

Share Document