Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.7.7 (DNA polymerase)
 17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A novel dNTP pyrophosphatase, Mj0226 from Methanococcus jannaschii, which catalyzes the hydrolysis of nucleoside triphosphates to the monophosphate and PPi, has been characterized. Mj0226 protein catalyzes hydrolysis of two major substrates, dITP and XTP, suggesting that the 6-keto group of hypoxanthine and xanthine is critical for interaction with the protein. Under optimal reaction conditions the k(ca)(t) /K(m) value for these substrates was approximately 10 000 times that with dATP. Neither endonuclease nor 3'-exonuclease activities were detected in this protein. Interestingly, dITP was efficiently inserted opposite a dC residue in a DNA template and four dNTPs were also incorporated opposite a hypoxanthine residue in template DNA by DNA polymerase I. Two protein homologs of Mj0226 from Escherichia coli and Archaeoglobus fulgidus were also cloned and purified. These have catalytic activities similar to Mj0226 protein under optimal conditions. The implications of these results have significance in understanding how homologous proteins, including Mj0226, act biologically in many organisms. It seems likely that Mj0226 and its homologs have a major role in preventing mutations caused by incorporation of dITP and XTP formed spontaneously in the nucleotide pool into DNA. This report is the first identification and functional characterization of an enzyme hydrolyzing non-canonical nucleotides, dITP and XTP.
 ...
PMID:Biochemical characterization of a novel hypoxanthine/xanthine dNTP pyrophosphatase from Methanococcus jannaschii. 1145 35

Spontaneous damage to DNA as a result of deamination, oxidation and depurination is greatly accelerated at high temperatures. Hyperthermophilic microorganisms constantly exposed to temperatures exceeding 80 degrees C are endowed with powerful DNA repair mechanisms to maintain genome stability. Of particular interest is the processing of DNA lesions during replication, which can result in fixed mutations. The hyperthermophilic crenarchaeon Sulfolobus solfataricus has two functional DNA polymerases, PolB1 and PolY1. We have found that the replicative DNA polymerase PolB1 specifically recognizes the presence of the deaminated bases hypoxanthine and uracil in the template by stalling DNA polymerization 3-4 bases upstream of these lesions and strongly associates with oligonucleotides containing them. PolB1 also stops at 8-oxoguanine and is unable to bypass an abasic site in the template. PolY1 belongs to the family of lesion bypass DNA polymerases and readily bypasses hypoxanthine, uracil and 8-oxoguanine, but not an abasic site, in the template. The specific recognition of deaminated bases by PolB1 may represent an initial step in their repair while PolY1 may be involved in damage tolerance at the replication fork. Additionally, we reveal that the deaminated bases can be introduced into DNA enzymatically, since both PolB1 and PolY1 are able to incorporate the aberrant DNA precursors dUTP and dITP.
 ...
PMID:Processing of DNA lesions by archaeal DNA polymerases from Sulfolobus solfataricus. 1285 19

The sequence saturation mutagenesis (SeSaM) method has been advanced to a random mutagenesis method with adjustable mutational biases. SeSaM offers, for example, a bias that is complementary to error-prone (ep) PCR and is enriched in transversions (SeSaM-Tv(+)). dNTP alpha S and three degenerate bases (P, K and I) are used to control mutational bias flexibly. After quantifying incorporation rates of dPTP, dKTP and dITP by terminal transferase using a luciferase-based assay and investigating the read and/or write activities of eight DNA polymerases, a transversion-enriched protocol has been developed. In a mutant library generated using dGTP alpha S and dPTP, transversion frequencies of 16.22-22.58% (G-->T) and 6.38-9.69% (G-->C) were achieved. These mutational spectra are complementary and occur twice as frequently in comparison to standard epPCR methods employing Taq DNA polymerase. For generating more complex mutant libraries, the occurrence of consecutive nucleotide exchanges was increased by 10(5)-10(6)-fold compared to epPCR. Finally, 16.7% of all sequenced mutants contained consecutive nucleotide exchanges composed mainly of a transversion followed by a transition.
 ...
PMID:Transversion-enriched sequence saturation mutagenesis (SeSaM-Tv+): a random mutagenesis method with consecutive nucleotide exchanges that complements the bias of error-prone PCR. 1802 59

The known archaeal family B DNA polymerases are unable to participate in the PCR in the presence of uracil. Here, we report on a novel archaeal family B DNA polymerase from Nanoarchaeum equitans that can successfully utilize deaminated bases such as uracil and hypoxanthine and on its application to PCR. N. equitans family B DNA polymerase (Neq DNA polymerase) produced lambda DNA fragments up to 10 kb with an approximately 2.2-fold-lower error rate (5.53 x 10(-6)) than Taq DNA polymerase (11.98 x 10(-6)). Uniquely, Neq DNA polymerase also amplified lambda DNA fragments using dUTP (in place of dTTP) or dITP (partially replaced with dGTP). To increase PCR efficiency, Taq and Neq DNA polymerases were mixed in different ratios; a ratio of 10:1 efficiently facilitated long PCR (20 kb). In the presence of dUTP, the PCR efficiency of the enzyme mixture was two- to threefold higher than that of either Taq and Neq DNA polymerase alone. These results suggest that Neq DNA polymerase and Neq plus DNA polymerase (a mixture of Taq and Neq DNA polymerases) are useful in DNA amplification and PCR-based applications, particularly in clinical diagnoses using uracil-DNA glycosylase.
 ...
PMID:Unique substrate spectrum and PCR application of Nanoarchaeum equitans family B DNA polymerase. 1879 Oct 30


<< Previous 1 2