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1.
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
2.
A novel strategy to engineer DNA polymerases for enhanced processivity and improved performance in vitro 总被引:9,自引:0,他引:9
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Wang Y Prosen DE Mei L Sullivan JC Finney M Vander Horn PB 《Nucleic acids research》2004,32(3):1197-1207
Mechanisms that allow replicative DNA polymerases to attain high processivity are often specific to a given polymerase and cannot be generalized to others. Here we report a protein engineering-based approach to significantly improve the processivity of DNA polymerases by covalently linking the polymerase domain to a sequence non-specific dsDNA binding protein. Using Sso7d from Sulfolobus solfataricus as the DNA binding protein, we demonstrate that the processivity of both family A and family B polymerases can be significantly enhanced. By introducing point mutations in Sso7d, we show that the dsDNA binding property of Sso7d is essential for the enhancement. We present evidence supporting two novel conclusions. First, the fusion of a heterologous dsDNA binding protein to a polymerase can increase processivity without compromising catalytic activity and enzyme stability. Second, polymerase processivity is limiting for the efficiency of PCR, such that the fusion enzymes exhibit profound advantages over unmodified enzymes in PCR applications. This technology has the potential to broadly improve the performance of nucleic acid modifying enzymes. 相似文献
3.
The replication DNA polymerase (gp43) of the bacteriophage T4 is a member of the pol B family of DNA polymerases, which are found in all divisions of life in the biosphere. The enzyme is a modularly organized protein that has several activities in one polypeptide chain (900 amino acid residues). These include two catalytic functions, POL (polymerase) and EXO (3-exonuclease), and specific binding activities to DNA, the mRNA for gp43, deoxyribonucleotides (dNTPs), and other T4 replication proteins. The gene for this multifunctional enzyme (gene 43) has been preserved in evolution of the diverse group of T4-like phages in nature, but has diverged in sequence, organization, and specificity of the binding functions of the gene product. We describe here examples of T4-like phages where DNA rearrangements have created split forms of gene 43 consisting of two cistrons instead of one. These gene 43 variants specify separate gp43A (N-terminal) and gp43B (C-terminal) subunits of a split form of gp43. Compared to the monocistronic form, the interruption in contiguity of the gene 43 reading frame maps in a highly diverged sequence separating the code for essential components of two major modules of this pol B enzyme, the FINGERS and PALM domains, which contain the dNTP binding pocket and POL catalytic residues of the enzyme. We discuss the biological implications of these gp43 splits and compare them to other types of pol B splits in nature. Our studies suggest that DNA mobile elements may allow genetic information for pol B modules to be exchanged between organisms.Translated from Biokhimiya, Vol. 69, No. 11, 2004, pp. 1489–1496.Original Russian Text Copyright © 2004 by Petrov, Karam. 相似文献
4.
Jun Yang Jing Yang Zheng-Qin Yin Jing Xu Ning Hu I. Svir Min Wang Yuan-Yi Li Lei Zhan Song Wu Xiao-Lin Zheng 《Biochemistry. Biokhimii?a》2009,74(7):813-818
The binding of human DNA polymerase β (pol β) to DNA template-primer duplex and single-stranded DNA in the absence or presence
of pol β inhibitors has been studied using a surface plasmon resonance biosensor. Two fatty acids, linoleic acid and nervonic
acid, were used as potent pol β inhibitors. In the interaction between pol β and DNA, pol β could bind to ssDNA in a single
binding mode, but bound to DNA template-primer duplexes in a parallel mode. Both pol β inhibitors prevented the binding of
pol β to the single strand overhang and changed the binding from parallel to single mode. The affinities of pol β to the template-primer
duplex region in the presence of nervonic acid or linoleic acid were decreased by 20 and 5 times, respectively. The significant
inhibitory effect of nervonic acid on the pol β-duplex interaction was due to both a 2-fold decrease in the association rate
and a 9-fold increase in the dissociation rate. In the presence of linoleic acid, no significant change of association rate
was observed, and the decrease in binding affinity of pol β to DNA was mainly due to 7-fold increase in the dissociation rate.
Published in Russian in Biokhimiya, 2009, Vol. 74, No. 7, pp. 1000–1006.
These authors contributed equally. 相似文献
5.
Geranylhydroquinone 3′′-hydroxylase, a cytochrome P-450 monooxygenase from Lithospermum erythrorhizon cell suspension cultures 总被引:2,自引:0,他引:2
Geranylhydroquinone 3′′-hydroxylase, which is likely to be involved in shikonin and dihydroechinofuran biosynthesis, was
identified in cell suspension cultures of Lithospermum erythrorhizon Sieb. et Zucc. (Boraginaceae). The enzyme hydroxylates the isoprenoid side chain of geranylhydroquinone (GHQ), a known precursor
of shikonin. Proton/proton correlation spectroscopic and proton/proton long-range correlation spectroscopic studies confirmed
that hydroxylation takes place specifically at position 3′′, i.e. at the methyl group involved in the cyclization reaction.
The enzyme is membrane-bound and was found in the microsomal fraction. It requires NADPH and molecular oxygen as cofactors,
and is inhibited by cytochrome P-450 inhibitors such as cytochrome c and CO. The inhibitory effect of CO is reversed by illumination.
These data suggest that the enzyme is a cytochrome P-450-dependent monooxygenase. The optimum pH of GHQ 3′′-hydroxylase is
7.4, and the apparent K
m value for GHQ is 1.5 μM. The reaction velocity obtained with 3-geranyl-4-hydroxybenzoic acid was more than 100 times lower
than that obtained with geranylhydroquinone.
Received: 20 March 1999 / Accepted: 20 July 1999 相似文献
6.
Butylphenyl dGTP: a selective and potent inhibitor of mammalian DNA polymerase alpha. 总被引:18,自引:13,他引:5
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BuPdGTP , the 2'-deoxyribonucleoside 5'-triphosphate of the DNA polymerase alpha (pol alpha)-specific inhibitor, N2-(p-n- butylphenyl )guanine, was examined with respect to its mechanism and its capacity to inhibit the mammalian DNA polymerases, pol alpha, pol beta, and pol gamma. BuP dGTP was specifically inhibitory for pol alpha, with no discernible activity on pol beta and pol gamma. The potency of BuP dGTP is unprecedented, with an apparent Ki less than 10 nanomolar. The unusual potency of the BuP dGTP is derived primarily from the 5' alpha and beta phosphoryl moieties, whose binding to enzyme complements that of the base-linked butylphenyl substituent. BuP dGTP is competitive with dGTP and apparently not subject to polymerization. Experiments employing BuP dGTP in the presence of a non-complementary template suggest that the core polymerase or an associated coprotein contains dNTP binding sites which recognize specific nucleic acid bases. The partial sensitivity of selected, non-mammalian DNA polymerases suggests that modification of the N2 substituent of dGTP will be a useful route to the design of novel, polymerase-specific affinity-probes. 相似文献
7.
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
8.
R. J. Phillips D. C. Hickleton P. E. Boehmer P. T. Emmerson 《Molecular & general genetics : MGG》1997,254(3):319-329
To investigate the role that the individual subunits play in the ATP-dependent helicase activity of the RecBCD protein we
have investigated the ability of the RecB, RecC and RecD proteins to displace various 20-mer oligonucleotides annealed to
either end or to the centre of an oligonucleotide 60 bases long. The results show that the only subunit which can displace
the 20-mers in the absence of the other subunits is the RecB protein. Moreover, the 20-mer is displaced only if it is annealed
to the 60-mer at the 5′ end or the middle, suggesting that the RecB protein translocates along the 60-mer in the 3′ to 5′
direction, displacing annealed 20-mers as it proceeds. We have shown that reconstituted RecBC and RecBCD complexes displace
the 20-mers but, unlike RecB, they do not require a 3′-ended single-stranded region for helicase action, but can displace
the 20-mers from either end of the 60-mer. The level of helicase activity of the RecBC complex is considerably greater than
that of RecB alone, and the activity of the RecBCD complex appears to be greater still. This hierarchy of activity is also
shown by DNA binding studies, but is not reflected in the ATPase activities of the enzymes. We have also shown that the ability
of trypsin to cleave various sites on the RecB molecule is modified by the presence of ATP or ATP-γ-S, suggesting that conformational
changes may be induced in RecB upon ATP binding. We discuss a model for the ATP-driven, unidirectional motion of the RecB
translocase along single-stranded DNA. In this model, the RecB molecule binds to single-stranded DNA and then translocates
along it, one base at a time, in the 3′ to 5′ direction, by a `ratchet' mechanism in which repeated stretching and contraction
of the protein is coupled to ATP hydrolysis. The RecC protein in the RecBC complex is proposed to act as a `sliding clamp'
which increases processivity by preventing dissociation.
Received: 10 September 1996 / Accepted: 18 November 1996 相似文献
9.
Sso7d from the extreme thermophilic crenarchaeon Sulfolobus solfataricus is a multifunctional protein in in vitro assays, whose in vivo role is still puzzling. Crystals of Sso7d in complex with DNA elucidated the protein surface involved in the binding to the nucleic acid, whereas the locations of the Sso7d regions responsible for a chaperone activity in renaturing protein aggregates (i.e., the protein-binding surface and the site of ATPase activity) are still unknown. We identified the regions of Sso7d involved in protein-binding by limited proteolysis experiments associated to advanced mass spectrometric procedures performed on isolated Sso7d and Sso7d in complex with the peptide melittin. By affinity labeling of Sso7d with the ATP analogue 5'-p-fluorosulfonylbenzoyl adenosine and characterization of the labeled tryptic peptides by tandem mass spectrometry, we found that Y7 and K39 are residues involved in ATP binding/hydrolysis. Insights into the positions of the ligands melittin and ATP were achieved by a molecular modeling study; the models obtained were in agreement with most experimental data. A comparison among the complexes of Sso7d with DNA, with melittin, and with ATP showed that the DNA-binding surface and the protein-binding surface overlap, whereas the ATPase site is mostly independent of the binding sites for the nucleic acid and melittin. 相似文献
10.
Bacterial family C DNA polymerases (DNA pol IIIs), the major chromosomal replicative enzymes, have been provisionally classified
based on primary sequences and domain structures into three classes: class I (Escherichia coli DNA pol C-type), class II (Bacillus subtilis DNA pol C-type), and class III (cyanobacterial DNA pol C-type), respectively. We have sequenced the structural gene encoding
the DNA pol C catalytic subunit of the thermophilic bacterium Thermus aquaticus. This gene, designated the Taq DNA pol C gene, contains a 3660-bp open reading frame which specifies a polypeptide of molecular
weight of 137,388 daltons. Comparative sequence analyses revealed that Taq DNA pol C is a class I family C DNA polymerase.
The Taq DNA pol C is most closely related to the Deinococcus radiodurans DNA pol C. Although a phylogenetic tree based on the class I family C DNA pols is still in the provisional stage, some important
conclusion can be drawn. First, the high-G+C and the low-G+C Gram-positive bacteria are not monophyletic. Second, the low-G+C
Gram-positive bacteria contain multigenes of family C DNA pols (classes I and II). Third, the cyanobacterial family C DNA
pol, classified as class III because it is encoded by a split gene, forms a group with the high-G+C Gram-positive bacteria.
Received: 7 October 1998 / Accepted: 12 January 1999 相似文献
11.
Metal-mediated hydrolysis of phosphate esters is a common catalytic pathway in nucleic acid biochemistry. Two distinct models
are principally invoked in mechanistic discussions of these reactions for magnesium-dependent nuclease activation: namely,
the one-versus two-metal-ion pathways. The 3′-5′ exonuclease domain of the Klenow fragment of Escherichia coli DNA polymerase I is a paradigm for the two-metal-ion mechanism; however, this reaction model is principally based on structural
and kinetics experiments employing high concentrations of transition metal analogues and high concentrations of background
ammonium sulfate during doping experiments. This prompted us to re-evaluate the metal cofactor stoichiometry of the 3′-5′
exonuclease mechanism for the Klenow fragment by solution kinetics and isothermal titration calorimetry using the natural
Mg2+ cofactor and salt conditions. Both solution calorimetric and kinetics experiments strongly indicate binding of only one metal
ion to the exonuclease active site. Comparative studies with Mn2+ also indicate a requirement for one metal ion to effect 3′-5′ exonuclease activity.
Received, accepted: 16 March 1998 相似文献
12.
13.
Grúz P Pisani FM Shimizu M Yamada M Hayashi I Morikawa K Nohmi T 《The Journal of biological chemistry》2001,276(50):47394-47401
DNA replication efficiency is dictated by DNA polymerases (pol) and their associated proteins. The recent discovery of DNA polymerase Y family (DinB/UmuC/RAD30/REV1 superfamily) raises a question of whether the DNA polymerase activities are modified by accessory proteins such as proliferating cell nuclear antigen (PCNA). In fact, the activity of DNA pol IV (DinB) of Escherichia coli is enhanced upon interaction with the beta subunit, the processivity factor of DNA pol III. Here, we report the activity of Sso DNA pol Y1 encoded by the dbh gene of the archaeon Sulfolobus solfataricus is greatly enhanced by the presence of PCNA and replication factor C (RFC). Sso pol Y1 per se was a distributive enzyme but a substantial increase in the processivity was observed on poly(dA)-oligo(dT) in the presence of PCNA (039p or 048p) and RFC. The length of the synthesized DNA product reached at least 200 nucleotides. Sso pol Y1 displayed a higher affinity for DNA compared with pol IV of E. coli, suggesting that the two DNA polymerases have distinct reason(s) to require the processivity factors for efficient DNA synthesis. The abilities of pol Y1 and pol IV to bypass DNA lesions and their sensitive sites to protease are also discussed. 相似文献
14.
A single highly mutable catalytic site amino acid is critical for DNA polymerase fidelity 总被引:6,自引:0,他引:6
Patel PH Kawate H Adman E Ashbach M Loeb LA 《The Journal of biological chemistry》2001,276(7):5044-5051
DNA polymerases contain active sites that are structurally superimposable and conserved in amino acid sequence. To probe the biochemical and structure-function relationship of DNA polymerases, a large library (200,000 members) of mutant Thermus aquaticus DNA polymerase I (Taq pol I) was created containing random substitutions within a portion of the dNTP binding site (Motif A; amino acids 605-617), and a fraction of all selected active Taq pol I (291 out of 8000) was tested for base pairing fidelity; seven unique mutants that efficiently misincorporate bases and/or extend mismatched bases were identified and sequenced. These mutants all contain substitutions of one specific amino acid, Ile-614, which forms part of the hydrophobic pocket that binds the base and ribose portions of the incoming nucleotide. Mutant Taq pol Is containing hydrophilic substitution I614K exhibit 10-fold lower base misincorporation fidelity, as well as a high propensity to extend mispairs. In addition, these low fidelity mutants containing hydrophilic substitution for Ile-614 can bypass damaged templates that include an abasic site and vinyl chloride adduct ethenoA. During polymerase chain reaction, Taq pol I mutant I614K exhibits an error rate that is >20-fold higher relative to the wild-type enzyme and efficiently catalyzes both transition and transversion errors. These studies have generated polymerase chain reaction-proficient mutant polymerases containing substitutions within the active site that confers low base pairing fidelity and a high error rate. Considering the structural and sequence conservation of Motif A, it is likely that a similar substitution will yield active low fidelity DNA polymerases that are mutagenic. 相似文献
15.
16.
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
17.
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
18.
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
19.
The φ29-like phage genus of Podoviridae family contains phages B103, BS32, GA-1, M2, Nf, φ15, φ29, and PZA that all infect
Bacillus subtilis. They have very similar morphology and their genomes consist of linear double-stranded DNA of approximately 20 kb. The nucleotide
sequences of individual genomes or their parts determined thus far show that these phages evolved from a common ancestor.
A terminal protein (TP) that is covalently bound to the DNA 5′-end primes DNA replication of these phages. The same mechanism
of DNA replication is used by the Cp-1 related phages (also members of the Podoviridae family) and by the phage PRD1 (member
of the Tectoviridae family). Based on the complete or partial genomic sequence data of these phages it was possible to analyze
the evolutionary relationship within the φ29-like phage genus as well as to other protein-primed replicating phages. Noncoding
regions containing origins of replication were used in the analysis, as well as amino acid sequences of DNA polymerases, and
with the φ29-like phages also amino acid sequences of the terminal proteins and of the gene 17 protein product, an accessory
component of bacteriophage DNA replicating machinery. Included in the analysis are also results of a comparison of these phage
DNAs with the prophages present in the Bacillus subtilis genome. Based on this complex analysis we define and describe in more detail the evolutionary branches of φ29-like phages,
one branch consisting of phages BS32, φ15, φ29, and PZA, the second branch composed of phages B103, M2, and Nf, and the third
branch having phage GA-1 as its sole member. In addition, amino acid sequences of holins, proteins involved in phage lysis
were used to extend the evolutionary study to other phages infecting Gram-positive bacteria. The analysis based on the amino
acid sequences of holins showed several weak points in present bacteriophage classification.
Received: 14 April 1998 / Accepted: 31 July 1998 相似文献
20.
The thumb subdomain, located in various family B DNA polymerases in the C-terminal region, has been shown in their crystal structures to move upon binding of DNA, changing its conformation to nearly completely wrap around the DNA. It has therefore been involved in DNA binding. In agreement with this, partial proteolysis studies of 29 DNA polymerase have shown that the accessibility of the cleavage sites located in their C-terminal region is reduced in the presence of DNA or terminal protein (TP), indicating that a conformational change occurs in this region upon substrate binding and suggesting that this region might be involved in DNA and TP binding. Therefore, we have studied the role of the C-terminus of 29 DNA polymerase by deletion of the last 13 residues of this enzyme. This fragment includes a previously defined region conserved in family B DNA polymerases. The resulting DNA polymerase Δ13 was strongly affected in DNA binding, resulting in a distributive replication activity. Additionally, the capacity of the truncated polymerase to interact with TP was strongly reduced and its initiation activity was very low. On the other hand, its nucleotide binding affinity and its fidelity were not affected. We propose that the C-terminal 13 amino acids of 29 DNA polymerase are involved in DNA binding and in a stable interaction with the initiator protein TP, playing an important role in the intrinsic processivity of this enzyme during polymerization. 相似文献