共查询到20条相似文献,搜索用时 46 毫秒
1.
Fred W. Perrino Dan J. Mazur Heather Ward Scott Harvey 《Cell biochemistry and biophysics》1999,30(3):331-352
The polymerization of nucleotide analogs into DNA is a common strategy used to inhibit DNA synthesis in rapidly dividing tumor
cells and viruses. The mammalian DNA polymerases catalyze the insertion of the arabinofuranosyl analogs of dNTPs (aranucleotides)
into DNA efficiently, but elongate from the 3′ aranucleotides poorly. Slow elongation provides an opportunity for exonucleases
to remove aranucleotides. The exonuclease activity associated with DNA polymerase δ removes araCMP from 3′ termini with the
same efficiency that it removes a paired 3′ deoxycytosine suggesting that the proofreading exonucleases associated with DNA
polymerases might remove aranucleotides inefficiently. A separate 30 kDa exonuclease has been purified from mammalian cells
that removes araCMP from 3′ termini. The activity of this enzyme in the cell could remove aranucleotides from 3′ termini of
DNA and decrease the efficacy of the analogs. Inhibition analysis of the purified exonuclease shows that this enzyme is inhibited
by thioinosine monophosphate (TIMP) with aK
i=17 μM. When high TIMP levels are generated in HL-60 cells, incorporation of araC in DNA is increased about 16-fold relative to
total DNA synthesis. This increased araC in DNA is likely a result of exonuclease inhibition in the cell. Thus, exonucleases
in cells might play an important role in removing aranucleotides inserted by DNA polymerases. 相似文献
2.
It has been well known for decades that deoxyribonucleic acid (DNA) polymerases with proofreading function have a higher fidelity
in primer extension as compared to those without 3′ exonuclease activities. However, polymerases with proofreading function
have not been used in single nucleotide polymorphism (SNP) assays. Here, we describe a new method for single-base discrimination
by proofreading the 3′ phosphorothioate-modified primers using a polymerase with proofreading function. Our data show that
the combination of a polymerase with 3′ exonuclease activity and the 3′ phosphorothioate-modified primers work efficiently
as a single-base mismatch-operated on/off switch. DNA polymerization only occurred from matched primers, whereas mismatched
primers were not extended at the broad range of annealing temperature tested in our study. This novel single-base discrimination
method has potential in SNP assays. 相似文献
3.
Hilario E 《Molecular biotechnology》2004,28(1):77-80
There are two ways to label a DNA molecular; by the ends or all along the molecule. End labeling can be performed at the 3′-
or 5′-end. Labeling at the 3′ end is performed by filling 3′-end recessed ends with a mixture or labeled and unlabeled dNTPs
using Klenow or T4 DNA polymerases. Both reactions are template dependent. Terminal deoxynucleotide transferase incorporates
dNTPs at the 3′ end of any kind of DNA molecule or RNA. Labels incorporated at the 3′-end of the DNA molecule prevent any
further extension or ligation to any other molecule, but this can be overcome by labeling the 5′-end of the desired DNA molecule.
5′-end labeling is performed by enzymatic methods (T4 polynucleotide kinase exchange and forward reactions), by chemical modification
of sensitized oligonucleotides with phosphoroamidite, or by combined methods. Probe cleanup is recommended when high background
problems occur, but caution should be taken not to damage the attached probe with harsh chemicals or by light exposure. 相似文献
4.
An ORF of 1716 nucleotides, putatively encoding a DNA polymerase, was characterized in the mitochondrial genome of the edible
basidiomycete Agrocybe aegerita. The complete gene, named Aa-polB, and its flanking regions were cloned and sequenced from three overlapping restriction fragments. Aa-polB is located between the SSU rDNA (5′ region) and a gene for tRNAAsn (3′ region), and is separated from these genes by two A+T-rich intergenic regions of 1048 (5′ region) and 3864 (3′ region)
nucleotides, which lack repeated sequences of mitochondrial or plasmid origin. The deduced Aa-POLB protein shows extensive
sequence similarity with the family B DNA polymerases encoded by genomes that rely on protein-primed replication (invertrons).
The domains involved in the 3′→5′ exonuclease (Exo I to III) and polymerase (Pol I to Pol V) activities were localized on
the basis of conserved sequence motifs. The alignment of the Aa-POLB protein (571 amino acids) with sequences of family B
DNA polymerases from invertrons revealed that in Aa-POLB the N-terminal region preceding Exo I is short, suggesting a close
relationship with the DNA polymerases of bacteriophages that have linear DNA. The Aa-polB gene was shown to be present in all wild strains examined, which were collected from a wide range of locations in Europe.
As shown by RT-PCR, the Aa-polB gene is transcribed in the mitochondria, at a low but significant level. The likelihood of the coexistence of Aa-POLB and
Pol γ in the A. aegerita mitochondrion is discussed in the light of recent reports showing the conservation of the nucleus-encoded Pol γ from yeast
to human.
Received: 13 October 1998 / Accepted: 21 December 1998 相似文献
5.
There are many various chromosomal and gene mutations in human cancer cells. The total mutation rate in normal human cells
is 2·10−7 mutations/gene/division. From 6 to 12 carcinogenic mutations can arise by the end of the life, and these can affect the structure
of ∼150 protooncogenes and genes encoding suppressors of tumor growth. However, this does not explain the tens and hundreds
of thousands of mutations detectable in cancer cells. Mutation is any change of nucleotide sequence in cellular DNA. Gene
mutations are mainly consequences of errors of DNA polymerases, especially of their specialized fraction (inaccurate DNA polymerases
β, ζ, η, θ, ι, κ, λ, μ, σ, ν, Rev1, and terminal deoxynucleotidyl transferase, and only polymerases θ and σ manifest a slight
3′-exonuclease activity) and also consequences of a decrease in the rate of repair of these errors. Inaccurate specialized
human polymerases are able to synthesize DNA opposite lesions in the DNA template, but their accuracy is especially low during
synthesis on undamaged DNA. In the present review fundamental features of such polymerases are considered. DNA synthesis stops
in the area of its lesion, but this block is overcome due to activities of inaccurate specialized DNA polymerases. After the
lesion is bypassed, DNA synthesis is switched to accurate polymerases α, δ, ɛ, or γ. Mechanisms of direct and reverse switches
of DNA polymerases as well as their modifications during carcinogenesis are discussed. 相似文献
6.
N. L. Ronzhina T. P. Kravetskaya V. M. Krutyakov 《Journal of Evolutionary Biochemistry and Physiology》2000,36(3):262-266
Using methods of gel filtration and ultracentrifugation, cell-free extracts from 12 objects representing the main vertebrate
representatives (bony fish, amphibian, reptiles, birds, mammals, including human) were studied. The enzyme activity of autonomous
3′→5′-exonucleases (AE) has been established to be 25–90% of the total 3′→5′ exonuclease activity of the extracts. A part
of the AE is revealed in a zone of the DNA polymerases of the α-family and can be separated by changing Chromatographic conditions
or by repeated fractionation. The high activity of AE allows suggesting their substantial participation in the replicative
correction of the DNA-poly-merase errors as well as in the postreplicative correction of the heteroduplexes in the vertebrate
DNA. 相似文献
7.
Directional cloning of native PCR products with preformed sticky ends (Autosticky PCR) 总被引:1,自引:0,他引:1
A novel method for the directional cloning of native PCR products was developed. Abasic sites in DNA templates make DNA polymerases
stall at the site during synthesis of the complementary strand. Since the 5′ ends of PCR product strands contain built-in
amplification primers, abasic sites within the primers result in the formation of 5′ single-stranded overhangs at the ends
of the PCR product, enabling its direct ligation to a suitably cleaved cloning vector without any further modification. This
“autosticky PCR” (AS-PCR) overcomes the problems caused by end sensitivity of restriction enzymes, or internal restriction
sites within the amplified sequences, and enables the generation of essentially any desired 5′ overhang.
Received: 11 August 1998 / Accepted: 2 October 1998 相似文献
8.
F. M. Pisani Mariarita De Felice Giuseppe Manco Mosè Rossi 《Extremophiles : life under extreme conditions》1998,2(3):171-177
DNA polymerase from Sulfolobus solfataricus, strain MT4 (Sso DNA pol), was one of the first archaeal DNA polymerases to be isolated and characterized. Its encoding gene
was cloned and sequenced, indicating that Sso DNA pol belongs to family B of DNA polymerases. By limited proteolysis experiments
carried out on the recombinant homogeneous protein, we were able to demonstrate that the enzyme has a modular organization
of its associated catalytic functions (DNA polymerase and 3′-5′ exonuclease). Indeed, the synthetic function was ascribed
to the enzyme C-terminal portion, whereas the N-terminal half was found to be responsible for the exonucleolytic activity.
In addition, partial proteolysis studies were utilized to map conformational changes on DNA binding by comparing the cleavage
map in the absence or presence of nucleic acid ligands. This analysis allowed us to identify two segments of the Sso DNA pol
amino acid chain affected by structural modifications following nucleic acid binding: region 1 and region 2, in the middle
and at the C-terminal end of the protein chain, respectively. Site-directed mutagenesis studies will be performed to better
investigate the role of these two protein segments in DNA substrate interaction.
Received: January 22, 1998 / Accepted: February 16, 1998 相似文献
9.
Influence of stereochemistry of the 3′-terminal phosphorothioate (PS)-modified primers was studied in a single base extension
(SBE) assay to evaluate any improvements in specificity. SBE reactions were catalyzed by members of the high fidelity Pfu family of DNA polymerases with (exo+) or without (exo−) 3′ → 5′ exonucleolytic activity. The diastereomerically pure PS-labeled
primers used in these studies were obtained either by the stereospecific chemical synthesis invented in our laboratory or
by the more conventional ion-exchange chromatographic method for separation of a mixture of diastereomers (RP and SP). When the SBE reaction was performed in the presence of mispaired 2′-deoxyribonucleoside triphosphates (dNTPs), the “racemic”
3′-phosphorothioate primer mixture resulted in a lower level of 3′ → 5′ exonuclease-mediated cleavage products in comparison
to the SBE reactions carried out with the corresponding unmodified primer. When the diastereomerically pure RP 3′-phosphorothioate primer was examined, the results were largely the same as for the racemic 3′-phosphorothioate primer
mixture. In contrast, a 3′-PS primer of SP configuration displayed significantly improved performance in the SBE reaction. This included the lack of 3′ → 5′ proofreading
products, less mispriming, and improved yield of incorporation of the correct nucleotide. 相似文献
10.
Moreland D. Gibbs Rosalind A. Reeves David Mandelman Qingli Mi Jun Lee Peter L. Bergquist 《Extremophiles : life under extreme conditions》2009,13(5):817-826
Thermus aquaticus DNA polymerase (Taq polymerase) made the polymerase chain reaction feasible and led to a paradigm shift in genomic analysis. Other Thermus polymerases were reported to have comparable performance in PCR and there was an analysis of their properties in the 1990s.
We re-evaluated our earlier phylogeny of Thermus species on the basis of 16S rDNA sequences and concluded that the genus could be divided into eight clades. We examined 22
representative isolates and isolated their DNA polymerase I genes. The eight most diverse polymerase genes were selected to
represent the eight clades and cloned into an expression vector coding for a His-tag. Six of the eight polymerases were expressed
so that there was sufficient protein for purification. The proteins were purified to homogeneity and examination of the biochemical
characteristics showed that although they were competent to perform PCR, none was as thermostable as commercially available
Taq polymerase; all had similar error-frequencies to Taq polymerase and all showed the expected 5′–3′ exonuclease activity. We conclude that the initial selection of T. aquaticus for DNA polymerase purification was a far-reaching and fortuitous choice but simple mutagenesis procedures on other Thermus-derived polymerases should provide comparable thermostability for the PCR reaction. 相似文献
11.
With the use of polymerases having 3′ to 5′ exonuclease activity and 3′ phosphorothioate-modified allelespecific primers,
we recently devised a SNP-operated on/off switch controlling DNA polymerization. One advantage of this novel on/off switch
is its adaptability to arrayed primer extension. To further expand its application in genetic analysis, the new on/off switch
was evaluated in discrimination of the match/mismatch status of single nucleotides upstream from the primer 3′ terminal. A
set of seven amplicons was developed with the templates differing from each other by a single nucleotide. Using this set of
amplicons, the new on/off switch was shown to be able to efficiently discriminate single nucleotide polymorphisms from the
primer 3′ terminus to the −6 position from the primer 3′ terminus. These data, illustrating the broad single nucleotide discrimination
ability of this novel on/off switch, explain why the SNP-operated on/off switch is powerful in SNP analysis, and also indicate
useful applications to genetic analysis additional to SNP assay. First, these data broaden the application of the novel on/off
switch in the analysis of mutations other than SNPs. Second, it raises a nucleotide-walking algorithm suitable for de novo array-based sequencing analysis. 相似文献
12.
Eukaryotic DNA polymerases in DNA replication and DNA repair 总被引:16,自引:0,他引:16
Peter M. J. Burgers 《Chromosoma》1998,107(4):218-227
DNA polymerases carry out a large variety of synthetic transactions during DNA replication, DNA recombination and DNA repair.
Substrates for DNA polymerases vary from single nucleotide gaps to kilobase size gaps and from relatively simple gapped structures
to complex replication forks in which two strands need to be replicated simultaneously. Consequently, one would expect the
cell to have developed a well-defined set of DNA polymerases with each one uniquely adapted for a specific pathway. And to
some degree this turns out to be the case. However, in addition we seem to find a large degree of cross-functionality of DNA
polymerases in these different pathways. DNA polymerase α is almost exclusively required for the initiation of DNA replication
and the priming of Okazaki fragments during elongation. In most organisms no specific repair role beyond that of checkpoint
control has been assigned to this enzyme. DNA polymerase δ functions as a dimer and, therefore, may be responsible for both
leading and lagging strand DNA replication. In addition, this enzyme is required for mismatch repair and, together with DNA
polymerase ζ, for mutagenesis. The function of DNA polymerase ɛ in DNA replication may be restricted to that of Okazaki fragment
maturation. In contrast, either polymerase δ or ɛ suffices for the repair of UV-induced damage. The role of DNA polymerase
β in base-excision repair is well established for mammalian systems, but in yeast, DNA polymerase δ appears to fullfill that
function.
Received: 20 April 1998 / Accepted: 8 May 1998 相似文献
13.
Cellular DNA polymerases belong to several families and carry out different functions. Highly accurate replicative DNA polymerases
play the major role in cell genome replication. A number of new specialized DNA polymerases were discovered at the turn of
XX–XXI centuries and have been intensively studied during the last decade. Due to the special structure of the active site,
these enzymes efficiently perform synthesis on damaged DNA but are characterized by low fidelity. Human DNA polymerase iota
(Pol ι) belongs to the Y-family of specialized DNA polymerases and is one of the most error-prone enzymes involved in DNA
synthesis. In contrast to other DNA polymerases, Pol ι is able to use noncanonical Hoogsteen interactions for nucleotide base
pairing. This allows it to incorporate nucleotides opposite various lesions in the DNA template that impair Watson-Crick interactions.
Based on the data of X-ray structural analysis of Pol ι in complexes with various DNA templates and dNTP substrates, we consider
the structural peculiarities of the Pol ι active site and discuss possible mechanisms that ensure the unique behavior of the
enzyme on damaged and undamaged DNA. 相似文献
14.
15.
This report describes a novel and efficient method for walking the sequence of a genomic deoxyribonucleic acid (DNA) from
a known region to an unknown region based on an oligodeoxynucleotide (oligo) cassette-mediated polymerase chain reaction technique.
In this method, genomic DNA is digested by a restriction enzyme that generates a sticky 5′-end, followed by ligation of a
one-base excess oligo-adaptor using T4 DNA ligase. The adaptor consists of two complementary oligos that form the same sticky
end as the digested genomic DNA fragments, except that the 5′-overhang base overlaps the corresponding 3′-end base of the
restriction site. This overhanging terminal base prevents ligation between the adaptors, and the appropriate molar ratio of
adaptor to genomic DNA enables specific amplification of the target sequence. T4 DNA ligase catalyzes both the ligation of
the phosphorylated overhang base of the adaptor to genomic DNA and the excision of the corresponding 3′-terminal base of the
genomic DNA. This sequence-specific exonuclease activity of T4 DNA ligase was confirmed by ligation of an alternative adaptor
in which the 5′-terminal base was not consistent with the corresponding 3′-terminal base. Using this technique, the 3′- and
5′-flanking sequences of the catalase gene of the ciliate Paramecium bursaria were determined. 相似文献
16.
De Felice M Medagli B Esposito L De Falco M Pucci B Rossi M Grùz P Nohmi T Pisani FM 《Extremophiles : life under extreme conditions》2007,11(2):277-282
The hyper-thermophilic archaeon Sulfolobus solfataricus possesses two functional DNA polymerases belonging to the B-family (Sso DNA pol B1) and to the Y-family (Sso DNA pol Y1).
Sso DNA pol B1 recognizes the presence of uracil and hypoxanthine in the template strand and stalls synthesis 3–4 bases upstream
of this lesion (“read-ahead” function). On the other hand, Sso DNA pol Y1 is able to synthesize across these and other lesions
on the template strand. Herein we report evidence that Sso DNA pol B1 physically interacts with DNA pol Y1 by surface plasmon
resonance measurements and immuno-precipitation experiments. The region of DNA pol B1 responsible for this interaction has
been mapped in the central portion of the polypeptide chain (from the amino acid residue 482 to 617), which includes an extended
protease hyper-sensitive linker between the N- and C-terminal modules (amino acid residues Asn482-Ala497) and the α-helices
forming the “fingers” sub-domain (α-helices R, R′ and S). These results have important implications for understanding the polymerase-switching mechanism on the damaged template
strand during genome replication in S. solfataricus. 相似文献
17.
Some drugs are routinely prescribed for dementia that sets in either due to normal ageing or due to neurodegenerative disorders.
We have studied the effect of three of these drugs, Donepezil hydrochloride, Rivastigmine tartrate and Nootropyl, on the activity
of DNA polymerases β, a crucial enzyme in the base excision repair pathway, the most important mode of DNA repair in brain.
All the three drugs inhibited DNA polymerase β activity to varying degrees although the affects of Donepezil being the least
and inconsistent. The drugs preferentially bind to and inhibit the activities of 8 kDa domain of DNA polymerase β that is
known to possess the dRP lyase activity. The function of 31 kDa domain dealing with template driven addition of nucleotides
at 3′ end of the primer is not adversely affected. The inhibitory action of most widely used dementia drugs on DNA repair
potential signifies that pharma sector needs to consider this aspect especially while designing drugs targeted towards brain.
N. S. Chary—On project assignment from J.J College of Arts and Science, Tiruchirapally, Tamilnadu, India-620024. 相似文献
18.
Lahja Uitto Jussi Halleeen P?ivi Remes Tapio Kesti Juhani E. Syv?oja 《Chromosoma》1992,102(Z1):S142-S146
The 3′→5′ exonuclease activity of highly purified large form of human DNA polymerase epsilon was studied. The activity removes
mononucleotides from the 3′ end of an oligonucleotide with a non-processive mechanism and leaves 5′-terminal trinucleotide
non-hydrolyzed. This is the case both with single-stranded oligonucleotides and with oligonucleotides annealed to complementary
regions of M13DNA. However, the reaction rates with single-stranded oligonucleotides are at least ten-fold when compared to
those with completely base-paired oligonucleotides. Conceivably, mismatched 3′ end of an oligonucleotide annealed to M13DNA
is rapidly removed and the hydrolysis is slown down when double-stranded region is reached. The preferential removal of a
non-complementary 3′ end and the non-processive mechanism are consistent with anticipated proofreading function. In addition
to the 3′→5′ exonuclease activity, an 5′→3′ exonuclease activity is often present even in relatively highly purified DNA polymerase
epsilon preparates suggesting that such an activity may be an essential com-ponent for the action of this enzymein vivo. Contrary to the 3′→5′ exonuclease activity, the 5′→3′ exonuclease is separable from the polymerase activity. 相似文献
19.
Masaharu Takemura 《Journal of molecular evolution》2001,52(5):419-425
A number of molecular forms of DNA polymerases have been reported to be involved in eukaryotic nuclear DNA replication, with
contributions from α-, δ-, and ε-polymerases. It has been reported that δ-polymerase possessed a central role in DNA replication
in archaea, whose ancestry are thought to be closely related to the ancestor of eukaryotes. Indeed, in vitro experiment shown
here suggests that δ-polymerase has the potential ability to start DNA synthesis immediately after RNA primer synthesis. Therefore,
the question arises, where did the α-polymerase come from? Phylogenetic analysis based on the nucleotide sequence of several
conserved regions reveals that two poxviruses, vaccinia and variola viruses, have polymerases similar to eukaryotic α-polymerase
rather than δ-polymerase, while adenovirus, herpes family viruses, and archaeotes have eukaryotic δ-like polymerases, suggesting
that the eukaryotic α-polymerase gene is derived from a poxvirus-like organism, which had some eukaryote-like characteristics.
Furthermore, the poxvirus's proliferation independent from the host-cell nucleus suggests the possibility that this virus
could infect non-nucleated cells, such as ancestral eukaryotes. I wish to propose here a new hypothesis for the origin of
the eukaryotic nucleus, posing symbiotic contact of an orthopoxvirus ancestor with an archaebacterium, whose genome already
had a δ-like polymerase gene.
Received: 26 October 2000 / Accepted: 16 January 2001 相似文献
20.
Huiling Hao Yunquan Jiang S. J. Zhang Peng Zhang Rong X. Zeng Marietta Y. W. T. Lee 《Chromosoma》1992,102(Z1):S121-S127
A continuing theme of our laboraory, has been the understanding of human DNA polymerases at the structural level. We have
purified DNA polymerases delta, epsilon and alpha from human placenta. Monoclonal antibodies to these polymerases were isolated
and used as tools to study their immunochemical relationships. These studies have shown that while DNA polymerases delta,
epsilon and alpha are discrete protiens, they must share common structural features by virtue of the ability of several of
our monoclonal antibodies to exhibit cross-reactivity. A second approach we have taken is the molecular cloning of human DNA
polymerase delta and epsilon. We have cloned the DNA polymerase delta cDNA, and this has allowed us to compare its primary
structure to those of human polymerase alpha and other members of this polymerase family. Multiple sequence alignments have
revealed that human DNA polymerase delta is also closely related to the herpes virus family of DNA polymerases. In situ hybridization
has shown that the human DNA polymerase delta gene is localized to chromosome 19 q13.3–q13.4. In order to further determine
the functional regions of the DNA polymerase δ structure we are currently expressing human pol δ inE. coli and baculovirus systems. Other work in our laboratory is directed toward examining the expression of DNA polymerase δ during
the cell cycle. 相似文献