scholarly journals The Glutamate Effect on the Functionality of Pol I DNA Polymerases

2012 ◽  
Vol 102 (3) ◽  
pp. 282a
Author(s):  
Mytrang H. Do ◽  
Hiromi S. Brown ◽  
Vince J. LiCata
Keyword(s):  
Dna Polymerases ◽  
Pol I

scholarly journals Two Family B DNA Polymerases from Aeropyrum pernix, an Aerobic Hyperthermophilic Crenarchaeote

1999 ◽  
Vol 181 (19) ◽  
pp. 5984-5992 ◽  
Author(s):  
Isaac K. O. Cann ◽  
Sonoko Ishino ◽  
Norimichi Nomura ◽  
Yoshihiko Sako ◽  
Yoshizumi Ishino
Keyword(s):  
Amino Acid ◽  
Dna Polymerase ◽  
Dna Polymerases ◽  
Amino Acid Sequences ◽  
Pol Ii ◽  
Aeropyrum Pernix ◽  
Heat Stable ◽  
Pol I ◽  
Group I ◽  
Fraction I

ABSTRACT DNA polymerase activities in fractionated cell extract ofAeropyrum pernix, a hyperthermophilic crenarchaeote, were investigated. Aphidicolin-sensitive (fraction I) and aphidicolin-resistant (fraction II) activities were detected. The activity in fraction I was more heat stable than that in fraction II. Two different genes (polA and polB) encoding family B DNA polymerases were cloned from the organism by PCR using degenerated primers based on the two conserved motifs (motif A and B). The deduced amino acid sequences from their entire coding regions contained all of the motifs identified in family B DNA polymerases for 3′→5′ exonuclease and polymerase activities. The product ofpolA gene (Pol I) was aphidicolin resistant and heat stable up to 80°C. In contrast, the product of polB gene (Pol II) was aphidicolin sensitive and stable at 95°C. These properties of Pol I and Pol II are similar to those of fractions II and I, respectively, and moreover, those of Pol I and Pol II ofPyrodictium occultum. The deduced amino acid sequence ofA. pernix Pol I exhibited the highest identities to archaeal family B DNA polymerase homologs found only in the crenarchaeotes (group I), while Pol II exhibited identities to homologs found in both euryarchaeotes and crenarchaeotes (group II). These results provide further evidence that the subdomainCrenarchaeota has two family B DNA polymerases. Furthermore, at least two DNA polymerases work in the crenarchaeal cells, as found in euryarchaeotes, which contain one family B DNA polymerase and one heterodimeric DNA polymerase of a novel family.

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 Accuracy, lesion bypass, strand displacement and translocation by DNA polymerases

2004 ◽  
Vol 359 (1441) ◽  
pp. 17-23 ◽  
Author(s):  
Thomas A. Steitz ◽  
Y. Whitney Yin
Keyword(s):  
Conformational Change ◽  
Dna Polymerase ◽  
Active Site ◽  
Dna Polymerases ◽  
Lesion Bypass ◽  
Dna Structures ◽  
Template Strand ◽  
Pol I ◽  
Deoxynucleoside Triphosphate ◽  
Copy Dna

The structures of DNA polymerases from different families show common features and significant differences that shed light on the ability of these enzymes to accurately copy DNA and translocate. The structure of a B family DNA polymerase from phage RB69 exhibits an active–site closing conformational change in the fingers domain upon forming a ternary complex with primer template in deoxynucleoside triphosphate. The rotation of the fingers domain α–helices by 60° upon dNTP binding is analogous to the changes seen in other families of polymerases. When the 3' terminus is bound to the editing 3' exonuclease active site, the orientation of the DNA helix axis changes by 40° and the thumb domain re–orients with the DNA. Structures of substrate and product complexes of T7 RNA polymerase, a structural homologue of T7 DNA polymerase, show that family polymerases use the rotation conformational change of the fingers domain to translocate down the DNA. The fingers opening rotation that results in translocation is powered by the release of the product pyrophosphate and also enables the Pol I family polymerases to function as a helicase in displacing the downstream non–template strand from the template strand.

scholarly journals Stem-Loop Silencing Reveals that a Third Mitochondrial DNA Polymerase, POLID, Is Required for Kinetoplast DNA Replication in Trypanosomes

2008 ◽  
Vol 7 (12) ◽  
pp. 2141-2146 ◽  
Author(s):  
Julian Chandler ◽  
Anthula V. Vandoros ◽  
Brian Mozeleski ◽  
Michele M. Klingbeil
Keyword(s):  
Mitochondrial Dna ◽  
Dna Polymerases ◽  
Rapid Decline ◽  
Kinetoplast Dna ◽  
Stem Loop ◽  
Bacterial Dna ◽  
Progressive Loss ◽  
Pol I ◽  
Mitochondrial Dna Polymerase ◽  
Replication Intermediates

ABSTRACT Kinetoplast DNA (kDNA), the mitochondrial genome of trypanosomes, is a catenated network containing thousands of minicircles and tens of maxicircles. The topological complexity dictates some unusual features including a topoisomerase-mediated release-and-reattachment mechanism for minicircle replication and at least six mitochondrial DNA polymerases (Pols) for kDNA transactions. Previously, we identified four family A DNA Pols from Trypanosoma brucei with similarity to bacterial DNA Pol I and demonstrated that two (POLIB and POLIC) were essential for maintaining the kDNA network, while POLIA was not. Here, we used RNA interference to investigate the function of POLID in procyclic T. brucei. Stem-loop silencing of POLID resulted in growth arrest and the progressive loss of the kDNA network. Additional defects in kDNA replication included a rapid decline in minicircle and maxicircle abundance and a transient accumulation of minicircle replication intermediates before loss of the kDNA network. These results demonstrate that POLID is a third essential DNA Pol required for kDNA replication. While other eukaryotes utilize a single DNA Pol (Pol γ) for replication of mitochondrial DNA, T. brucei requires at least three to maintain the complex kDNA network.

scholarly journals Water Release upon DNA Binding by E. coli and T. aquaticus Pol I DNA Polymerases

2009 ◽  
Vol 23 (S1) ◽  
Author(s):  
John T. Baker ◽  
Daniel J. Deredge ◽  
Kausiki Datta ◽  
Vince J. LiCata
Keyword(s):  
Dna Binding ◽  
Dna Polymerases ◽  
Water Release ◽  
E Coli ◽  
Pol I
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