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1.
Porcine liver DNA polymerase gamma was shown previously to copurify with an associated 3' to 5' exonuclease activity (Kunkel, T. A., and Mosbaugh, D. W. (1989) Biochemistry 28, 988-995). The 3' to 5' exonuclease has now been characterized, and like the DNA polymerase activity, it has an absolute requirement for a divalent metal cation (Mg2+ or Mn2+), a relatively high NaCl and KCl optimum (150-200 mM), and an alkaline pH optimum between 7 and 10. The exonuclease has a 7.5-fold preference for single-stranded over double-stranded DNA, but it cannot excise 3'-terminal dideoxy-NMP residues from either substrate. Excision of 3'-terminally mismatched nucleotides was preferred approximately 5-fold over matched 3' termini, and the hydrolysis product from both was a deoxyribonucleoside 5'-monophosphate. The kinetics of 3'-terminal excision were measured at a single site on M13mp2 DNA for each of the 16 possible matched and mismatched primer.template combinations. As defined by the substrate specificity constant (Vmax/Km), each of the 12 mismatched substrates was preferred over the four matched substrates (A.T, T.A, C.G, G.C). Furthermore, the exonuclease could efficiently excise internally mismatched nucleotides up to 4 residues from the 3' end. DNA polymerase gamma was not found to possess detectable DNA primase, endonuclease, 5' to 3' exonuclease, RNase, or RNase H activities. The DNA polymerase and exonuclease activities exhibited dissimilar rates of heat inactivation and sensitivity to N-ethylmaleimide. After nondenaturing activity gel electrophoresis, the DNA polymerase and 3' to 5' exonuclease activities were partially resolved and detected in situ as separate species. A similar analysis on a denaturing activity gel identified catalytic polypeptides with molecular weights of 127,000, 60,000, and 32,000 which possessed only DNA polymerase gamma activity. Collectively, these results suggest that the polymerase and exonuclease activities reside in separate polypeptides, which could be derived from separate gene products or from proteolysis of a single gene product. 相似文献
2.
Recent crystallographic resolution of ?29 DNA polymerase complexes with ssDNA at its 3′-5′ exonuclease active site has allowed the identification of residues Pro129 and Tyr148 as putative ssDNA ligands, the latter being conserved in the Kx2h motif of proofreading family B DNA polymerases. Single substitution of ?29 DNA polymerase residue Tyr148 to Ala rendered an enzyme with a reduced capacity to stabilize the binding of the primer terminus at the 3′-5′ exonuclease active site, not having a direct role in the catalysis of the reaction. Analysis of the 3′-5′ exonuclease on primer/template structures showed a critical role for residue Tyr148 in the proofreading of DNA polymerisation errors. In addition, Tyr148 is not involved in coupling polymerisation to strand displacement in contrast to the catalytic residues responsible for the exonuclease reaction, its role being restricted to stabilisation of the frayed 3′ terminus at the exonuclease active site. Altogether, the results lead us to extend the consensus sequence of the above motif of proofreading family B DNA polymerases into Kx2hxA. The different solutions adopted by proofreading DNA polymerases to stack the 3′ terminus at the exonuclease site are discussed. In addition, the results obtained with mutants at ?29 DNA polymerase residue Pro129 allow us to rule out a functional role as ssDNA ligand for this residue. 相似文献
3.
Henry J. Russell Tomas T. Richardson Kieran Emptage Bernard A. Connolly 《Nucleic acids research》2009,37(22):7603-7611
Archaeal family B polymerases bind tightly to the deaminated bases uracil and hypoxanthine in single-stranded DNA, stalling replication on encountering these pro-mutagenic deoxynucleosides four steps ahead of the primer–template junction. When uracil is specifically bound, the polymerase–DNA complex exists in the editing rather than the polymerization conformation, despite the duplex region of the primer-template being perfectly base-paired. In this article, the interplay between the 3′–5′ proofreading exonuclease activity and binding of uracil/hypoxanthine is addressed, using the family-B DNA polymerase from Pyrococcus furiosus. When uracil/hypoxanthine is bound four bases ahead of the primer–template junction (+4 position), both the polymerase and the exonuclease are inhibited, profoundly for the polymerase activity. However, if the polymerase approaches closer to the deaminated bases, locating it at +3, +2, +1 or even 0 (paired with the extreme 3′ base in the primer), the exonuclease activity is strongly stimulated. In these situations, the exonuclease activity is actually stronger than that seen with mismatched primer-templates, even though the deaminated base-containing primer-templates are correctly base-paired. The resulting exonucleolytic degradation of the primer serves to move the uracil/hypoxanthine away from the primer–template junction, restoring the stalling position to +4. Thus the 3′–5′ proofreading exonuclease contributes to the inability of the polymerase to replicate beyond deaminated bases. 相似文献
4.
Response of human REV1 to different DNA damage: preferential dCMP insertion opposite the lesion 总被引:8,自引:6,他引:2
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Zhang Y Wu X Rechkoblit O Geacintov NE Taylor JS Wang Z 《Nucleic acids research》2002,30(7):1630-1638
REV1 functions in the DNA polymerase ζ mutagenesis pathway. To help understand the role of REV1 in lesion bypass, we have examined activities of purified human REV1 opposite various template bases and several different DNA lesions. Lacking a 3′→5′ proofreading exonuclease activity, purified human REV1 exhibited a DNA polymerase activity on a repeating template G sequence, but catalyzed nucleotide insertion with 6-fold lower efficiency opposite a template A and 19–27-fold lower efficiency opposite a template T or C. Furthermore, dCMP insertion was greatly preferred regardless of the specific template base. Human REV1 inserted a dCMP efficiently opposite a template 8-oxoguanine, (+)-trans-anti-benzo[a]pyrene-N 2-dG, (–)-trans-anti-benzo[a]pyrene-N 2-dG and 1,N 6-ethenoadenine adducts, very inefficiently opposite an acetylaminofluorene-adducted guanine, but was unresponsive to a template TT dimer or TT (6–4) photoproduct. Surprisingly, the REV1 specificity of nucleotide insertion was very similar in response to different DNA lesions with greatly preferred C insertion and least frequent A insertion. By combining the dCMP insertion activity of human REV1 with the extension synthesis activity of human polymerase κ, bypass of the trans-anti-benzo[a]pyrene-N 2 -dG adducts and the 1,N 6-ethenoadenine lesion was achieved by the two-polymerase two-step mechanism. These results suggest that human REV1 is a specialized DNA polymerase that may contribute to dCMP insertion opposite many types of DNA damage during lesion bypass. 相似文献
5.
N V Beliakova N E Kle?ner T P Kravetskaia O K Legina S N Naryzhny? F V Perrino I V Shevelev V M Krutiakov 《Izvestiia Akademii nauk SSSR. Seriia biologicheskaia》1992,(5):744-752
Mammalian nuclear DNA polymerases alpha and beta are lack of the proofreading 3'-->5' exonucleolytic activity. 40 and 50 kDa 3'-->5' exonucleases were isolated from rat liver. The exonucleases were shown to excise mismatched nucleotides from poly[d(A--T)] template 10 and 2 fold faster than matched ones. The addition of either exonuclease to DNA polymerase alpha from rat liver or calf thymus 5-10 times increased the accuracy of reproduction of primed DNA from bacteriophage phi X174 amber 3, values of exonuclease and DNA polymerase activities being approximately equal. The exonuclease activity surpasses the DNA polymerase one by an order of magnitude in chromatin and nuclear membrane. These data, taken together, are indicative of potent proofreading into hepatocytes. 相似文献
6.
Alfonso Jiménez-Sánchez 《Molecular & general genetics : MGG》1976,145(2):113-117
Summary Both the polymerase and the exonuclease activities of DNA polymerase III* are inactivated by treatment with nitrosoguanidine. The treatment of the DNA template with the mutagen does not affect the template in supporting DNA synthesis. No effect of nitrosoguanidine upon fidelity of replication in vitro was detected. 相似文献
7.
The Family X DNA Polymerase from Deinococcus radiodurans Adopts
a Non-standard Extended Conformation
Nicolas Leulliot Lionel Cladière Fran?ois Lecointe Dominique Durand Ulrich Hübscher Herman van Tilbeurgh 《The Journal of biological chemistry》2009,284(18):11992-11999
Deinococcus radiodurans is an extraordinarily radioresistant
bacterium that is able to repair hundreds of radiation-induced double-stranded
DNA breaks. One of the players in this pathway is an X family DNA polymerase
(PolXDr). Deletion of PolXDr has been shown to decrease
the rate of repair of double-stranded DNA breaks and increase cell sensitivity
to gamma-rays. A 3′→5′ exonuclease activity that stops
cutting close to DNA loops has also been demonstrated. The present crystal
structure of PolXDr solved at 2.46-Å resolution reveals that
PolXDr has a novel extended conformation in stark contrast to the
closed “right hand” conformation commonly observed for DNA
polymerases. This extended conformation is stabilized by the C-terminal PHP
domain, whose putative nuclease active site is obstructed by its interaction
with the polymerase domain. The overall conformation and the presence of non
standard residues in the active site of the polymerase X domain makes
PolXDr the founding member of a novel class of polymerases involved
in DNA repair but whose detailed mode of action still remains enigmatic.DNA replication and repair are functions that are of vital importance for
the maintenance of cellular life. These functions are carried out by various
DNA replicating engines, most of them acting as multiprotein complexes.
Deinococcus radiodurans, a Gram-positive bacterium, is characterized
by an extraordinary resistance to ionizing radiation and desiccation. After
radiation induced cutting of its 3.28-megabase genome into hundreds of small
fragments, it is capable of reassembling it completely
(1). Different hypotheses have
been suggested to explain this radioresistance. A recently proposed mechanism
involves the creation of long linear DNA intermediates by an extended
synthesis-dependent strand annealing process, where overlapping chromosomal
fragments are used both as primers and as templates for synthesis of
complementary single strands
(2). Recircularization of
chromosomes would be assured by homologous recombination. Although DNA
polymerase I is one of the main enzymes involved in this process, it was shown
that other proteins affect double strand break repair efficiency in D.
radiodurans. One of these is an X family DNA polymerase
(PolXDr)5
(3). Cells devoid of
PolXDr protein show increased sensitivity to γ-irradiation
and a longer delay in the restoration of an intact genome after irradiation.
It was therefore proposed that PolXDr has an important role in
double strand break repair in D. radiodurans. The contribution of
PolXDr may become essential for instance when damage gets too
important or, alternatively, it may act in different repair pathways from
polymerase I. Indeed, some of the X DNA polymerases, such as Saccharomyces
cerevisiae Pol4 and human polymerase λ
(4) have been proposed to play
important roles in different DNA repair processes, including non-homologous
end-joining (5). It was shown
that PolXDr also has strong 3′→5′ exonuclease
activity that is stimulated by Mn2+
(6). This activity is
associated with proofreading mechanisms in other polymerase families and
encoded by protein domains or subunits distinct from the polymerase catalytic
domain (7). Curiously the
exonuclease activity of PolXDr is modulated upon encounter of a
stem-loop structure. The combination of both activities leads to the
hypothesis that PolXDr might be involved in DNA repair, potentially
non-homologous end-joining, by processing damaged DNA or repair intermediates,
thus generating substrates for other repair proteins
(6). Very recently an
orthologue of PolX from Bacillus subtilis was characterized. It was
shown that PolXBs is a template-directed DNA polymerase acting on
DNA gaps with a downstream 5′ phosphate group, suggesting it may play a
role in base excision repair
(8).DNA polymerases all combine a catalytic palm domain, a thumb domain,
binding double-stranded DNA, and a finger domain that fixes the incoming
nucleotide. The polymerase domain of the X family belongs to the
Polβ-like nucleotidyltransferase superfamily, sharing ∼25% amino acid
identity with the DNA polymerase domains of Polλ, Pol4, and Polβ.
PolXDr has a second domain at the C terminus called PHP, with
strong sequence identity with the histidinol phosphatase involved in histidine
transport in bacteria. Due to its similarity to histidinol phosphatase and the
presence of a trinuclear zinc site, the PolXDr PHP domain is
thought to function as phosphoesterase
(9). In the context of DNA
polymerases, this activity might be responsible for the degradation of
pyrophosphate, thus driving the polymerization reaction, or contributes to a
nuclease reaction that would be involved in proofreading the newly synthesized
strand. The deletion of the PHP domain also had a negative effect on survival
of γ-irradiated cells suggesting that this domain possesses a function
in DNA repair. Unexpectedly, deletion of the PHP domain destroys structure
modulated but not the general 3′→5′ exonuclease activity
(6). No activity could be
demonstrated for the PHP domain alone.In this report we present the crystal structure of PolXDr at
2.46-Å resolution. Surprisingly, PolXDr adopts a stretched
out conformation instead of the commonly observed closed right hand
conformation. In the active site of the polymerase catalytic domain, the two
universally conserved aspartates are replaced by two glutamates, whereas the
active site of the PHP domain is obstructed by its interaction with the
polymerase domain. 相似文献
8.
The Klenow fragment of Escherichia coli DNA polymerase I houses catalytic centers for both polymerase and 3'-5' exonuclease activities that are separated by about 35 A. Upon the incorporation of a mismatched nucleotide, the primer terminus is transferred from the polymerase site to an exonuclease site designed for excision of the mismatched nucleotides. The structural comparison of the binary complexes of DNA polymerases in the polymerase and the exonuclease modes, together with a molecular modeling of the template strand overhang in Klenow fragment, indicated its binding in the region spanning residues 821-824. Since these residues are conserved in the "A" family DNA polymerases, we have designated this region as the RRRY motif. The alanine substitution of individual amino acid residues of this motif did not change the polymerase activity; however, the 3'-5' exonuclease activity was reduced 2-29-fold, depending upon the site of mutation. The R821A and R822A/Y824A mutant enzymes showed maximum cleavage defect with single-stranded DNA, mainly due to a large decrease in the ssDNA binding affinity of these enzymes. Mismatch removal by these enzymes was only moderately affected. However, data from the exonuclease-polymerase balance assays with mismatched template-primer suggest that the mutant enzymes are defective in switching mismatched primer from the polymerase to the exonuclease site. Thus, the RRRY motif provides a binding track for substrate ssDNA and for nonsubstrate single-stranded template overhang, in a polarity-dependent manner. This binding then facilitates cleavage of the substrate at the exonuclease site. 相似文献
9.
García Elpidio Orjuela Dairo Camacho Yolanda Zúñiga José Juan Plasencia Javier Vázquez-Ramos Jorge M. 《Plant molecular biology》1997,33(3):445-455
Three DNA polymerase activities, named 1, 2 and 3 were purified from maize embryo axes and were compared in terms of ion requirements, optimal pH, temperature and KCl for activity, response to specific inhibitors and use of templates. All three enzymes require a divalent cation for activity, but main differences were observed in sensitivity to inhibitors and template usage: while DNA polymerases 1 and 2 were inhibited by N-ethyl maleimide and aphidicolin, inhibitors of replicative-type enzymes, DNA polymerase 3 was only marginally or not affected at all. In contrast, DNA polymerase 3 was highly inhibited by very low concentrations of ddTTP, an inhibitor of repair-type enzymes, and a 100-fold higher concentration of the drug was needed to inhibit DNA polymerases 1 and 2. Additionally, DNA polymerases 1 and 2 used equally or more efficiently the synthetic template polydA-oligodT, as compared to activated DNA, while polymerase 3 used it very poorly. Whereas DNA polymerases 1 and 2 shared properties of replicative-type enzymes, DNA polymerase 3 could be a repair-type enzyme. Moreover, a DNA primase activity copurified with the 8000-fold purified DNA polymerase 2, strenghtening the suggestion that polymerase 2 is a replicative enzyme, of the -type. This DNA primase activity was also partially characterized. The results are discussed in terms of relevant data about other plant DNA polymerases and primases reported in the literature. 相似文献
10.
《Genetic analysis, techniques and applications》1998,14(3):75-83
We have cloned and sequenced the polA gene from Chloroflexus aurantiacus, a green nonsulfur eubacterium, and expressed the recombinant protein in Escherichia coli. One open reading frame encodes a protein with 942 amino acids showing 38% identity with DNA polymerase I from E. coli. Sequence alignments with other members of DNA polymerase family A and analysis of the separate domains show that the central 3′-5′ exonuclease domain is 30% identical to the corresponding E. coli domain and that three sequence motifs associated with 3′-5′ exonuclease activity are conserved. Also, a protein fraction from E. coli expressing the Chloroflexus polymerase contains a thermostable 3′-5′ exonucleolytic activity, indicating that this activity is present in the enzyme, in agreement with the sequence analysis. The N-terminal 5′-3′ exonuclease domain and the C-terminal polymerase domain show 31 and 46% identity, respectively, with the corresponding E. coli domains and all sequence motifs associated with these two enzymatic activities also are conserved. Since several DNA polymerase I enzymes lack the proofreading activity associated with the central domain it has been suggested that the ancestral polA gene contained only the two more conserved N- and C-terminal domains and that the proofreading 3′-5′ exonuclease domain was introduced later in those eubacterial branches that have this activity. Our data indicate a different scenario where the ancestral polA gene contained both the exonucleolytic activities in addition to the polymerase activity and where several eubacterial branches lost the polymerase-associated proofreading activity during evolution. 相似文献
11.
Escherichia coli exonuclease III enhances long PCR amplification of damaged DNA templates 总被引:1,自引:0,他引:1
Recent development of the long PCR technology has provided an invaluable tool in many areas of molecular biology. However, long PCR amplification fails whenever the DNA template is imperfectly preserved. We report that Escherichia coli exonuclease III, a major repair enzyme in bacteria, strikingly improves the long PCR amplification of damaged DNA templates. Escherichia coli exonuclease III permitted or improved long PCR amplification with DNA samples submitted to different in vitro treatments known to induce DNA strand breaks and/or apurinic/apyrimidinic (AP) sites, including high temperature (99°C), depurination at low pH and near-UV radiation. Exonuclease III also permitted or improved amplification with DNA samples that had been isolated several years ago by the phenol/chloroform method. Amelioration of long PCR amplification was achieved for PCR products ranging in size from 5 to 15.4 kb and with DNA target sequences located either within mitochondrial DNA or the nuclear genome. Exonuclease III increased the amplification of damaged templates using either rTth DNA polymerase alone or rTth plus Vent DNA polymerases or Taq plus Pwo DNA polymerases. However, exonuclease III could not improve PCR amplification from extensively damaged DNA samples. In conclusion, supplementation of long PCR mixes with E.coli exonuclease III may represent a major technical advance whenever DNA samples have been partly damaged during isolation or subsequent storage. 相似文献
12.
29 DNA polymerase achieves a functional coupling between its 3′–5′ exonuclease and polymerization activities by means of important contacts with the DNA at both active sites. The placement and orientation of residues Lys538, Lys555, Lys557, Gln560, Thr571, Thr573 and Lys575 in a modelled 29 DNA polymerase–DNA complex suggest a DNA-binding role. In addition, crystal structure of 29 DNA polymerase–oligo (dT)5 complex showed Leu567, placed at the tip of the thumb subdomain, lying between the two 3′-terminal bases at the exonuclease site. Single replacement of these 29 DNA polymerase residues by alanine was made, and mutant derivatives were overproduced and purified to homogeneity. The results obtained in the assay of their synthetic and degradative activities, as well as their coordination, allow us to propose: (1) a primer-terminus stabilization role at the polymerase active site for residues Lys538, Thr573 and Lys575, (2) a primer-terminus stabilization role at the exonuclease active site for residues Leu567 and Lys555 and (3) a primer-terminus binding role in both editing and polymerization modes for residue Gln560. The results presented here lead us to propose 29 DNA polymerase thumb as the main subdomain responsible for the coordination of polymerization and exonuclease activities. 相似文献
13.
Kausiki Datta Neil P. Johnson Vince J. LiCata Peter H. von Hippel 《The Journal of biological chemistry》2009,284(25):17180-17193
In addition to their capacity for template-directed 5′ → 3′ DNA synthesis at the polymerase (pol) site, DNA polymerases have a separate 3′ → 5′ exonuclease (exo) editing activity that is involved in assuring the fidelity of DNA replication. Upon misincorporation of an incorrect nucleotide residue, the 3′ terminus of the primer strand at the primer-template (P/T) junction is preferentially transferred to the exo site, where the faulty residue is excised, allowing the shortened primer to rebind to the template strand at the pol site and incorporate the correct dNTP. Here we describe the conformational changes that occur in the primer strand as it shuttles between the pol and exo sites of replication-competent Klenow and Klentaq DNA polymerase complexes in solution and use these conformational changes to measure the equilibrium distribution of the primer between these sites for P/T DNA constructs carrying both matched and mismatched primer termini. To this end, we have measured the fluorescence and circular dichroism spectra at wavelengths of >300 nm for conformational probes comprising pairs of 2-aminopurine bases site-specifically replacing adenine bases at various positions in the primer strand of P/T DNA constructs bound to DNA polymerases. Control experiments that compare primer conformations with available x-ray structures confirm the validity of this approach. These distributions and the conformational changes in the P/T DNA that occur during template-directed DNA synthesis in solution illuminate some of the mechanisms used by DNA polymerases to assure the fidelity of DNA synthesis.Escherichia coli DNA polymerase (DNAP)2 I is a single subunit polymerase that is organized into three functional domains: an N-terminal domain that is associated with 5′ → 3′ exonuclease activity, an intermediate domain that carries the 3′ → 5′ proofreading activity, and a C-terminal domain that is associated with the 5′ → 3′ template-directed polymerization activity. An important role of DNAP I is to remove the RNA primers of the Okazaki fragments formed during lagging strand DNA synthesis in E. coli replication and to fill in the resulting gaps by template-directed DNA synthesis (1). An N-terminal deletion mutant of DNAP I, known as the “large fragment” or Klenow form of the enzyme, contains only the polymerase (pol) and the 3′ → 5′ exonuclease (exo) domains. The Klenow polymerase has served and continues to serve as an excellent model system for isolating and defining general structure-function relationships in polymerases and in the supporting machinery of DNA replication.The main function of the 3′ → 5′ exonuclease activity of DNAP I is to remove misincorporated nucleotide residues from the 3′-end of the primer (2), thus contributing significantly to the overall fidelity of DNA replication (3). Contrary to initial expectations, crystallographic studies showed that the pol and exo active sites are quite far apart in replication polymerases, about 30 Å in Klenow (4). As a consequence, the ability of polymerases to “shuttle” the 3′-end of the primer strand efficiently between the pol and the exo sites in order to rectify misincorporation events during polymerization is critical to maintaining the overall accuracy of template-directed replication. Elucidation of the mechanisms of this shuttling and determination of the factors that control the rates (and equilibria) of the active site switching reaction will certainly increase our understanding of fidelity control by DNA polymerases.An early crystallographic study of the Klenow polymerase complexed with fully paired primer-template (P/T) DNA revealed that 3–4 nt of the 3′-primer terminus had been unwound from the template stand and partitioned into the exo site and that an extended single-stranded DNA (ssDNA) binding pocket of the exo site appeared to make position-specific hydrophobic contacts with the unstacked bases at the 3′-end of the primer (4). A separate crystallographic study of an editing complex confirmed that an ssDNA fragment 4 nt in length was bound at the exo site in the same conformation as seen for the single-stranded 3′-primer sequence unwound from P/T DNA (5). A structure of Klenow polymerase with the DNA bound at the pol site has not yet been reported, although such structures have been obtained for other homologous polymerases, including Klentaq (the “large fragment” of Thermus aquaticus (Taq) DNAP), Bacillus stearothermophilus (Bst) “large fragment” polymerase, and the T7 DNAP (6–8), all of which are members of the polymerase family that includes Klenow.The amino acid residues involved in the binding of DNA at the pol site in these polymerases (determined from co-crystal structures) and those of Klenow (determined by site-directed mutagenesis studies (9, 10)) are highly conserved, suggesting that a similar DNA binding mode at the pol site may apply to all of the DNAP I polymerases. The crystal structure of Klenow revealed that the polymerization domain has a shape reminiscent of a right hand in which the palm, fingers, and thumb domains form the DNA-binding crevice. Structural studies with various DNAP I polymerases in the presence of P/T DNA constructs yielded an “open” binary complex, whereas the addition of the next correct dNTP (as a chain-terminating dideoxy-NTP) resulted in the formation of a catalytically competent “closed” ternary complex (6–8). In the latter complex, the 3′-primer terminus was base-paired with the template DNA, and the templating base was poised for incorporation of the next correct nucleotide. These structures showed that the conformation of the DNA primer terminus bound at the pol site is markedly different from that of the “frayed open” primer observed at the exo site in Klenow (4, 5).Although crystallographic studies have provided a wealth of information about the conformations of the DNA substrates bound at the active sites of DNAP, replication itself is a dynamic process (reviewed in Ref. 11), and it is critical to be able to distinguish between various forms of DNA-polymerase complexes in solution in order to fully understand the mechanistic details of the replication process. A solution approach used by Millar and co-workers (reviewed in Ref. 12) for studying the conformation of DNA in these complexes involved measuring the time-resolved fluorescence anisotropy properties of a dansyl fluorophore attached to a DNA base located 8 bp upstream of the P/T DNA junction. The changes in the lifetime of the fluorophore, which appeared to depend mostly on the local environment occupied by the probe within the protein (i.e. buried versus partially exposed), were correlated with specific binding conformations of the primer to provide an estimate of the fractional occupancy of the pol and the exo sites. Reha-Krantz and co-workers (13) more recently used a related approach, here involving the monitoring of changes in the fluorescent lifetimes of a single 2-aminopurine (2-AP) base (a fluorescent analogue of adenine) site-specifically substituted in the template strand at the P/T junction, to make similar fractional occupancy measurements. However, we note that structural interpretations of these fluorescence experiments relied heavily on the available crystal structures, and it remained to be shown directly that the 3′-end of the primer in P/T DNA constructs assumes the same distribution of conformations when bound to the protein in solution.To get around this problem, as well as to directly investigate the conformations of the primer DNA in both active sites of the Klenow and Klentaq polymerases, we have used a novel CD spectroscopic approach to characterize the solution conformations of primer DNA bound to Klenow and Klentaq DNAPs. Previously, we had shown that CD spectroscopy, in conjunction with fluorescence measurements, can be used to examine changes in local DNA and RNA conformations at 2-AP dimer probes inserted at specified positions within the nucleic acid frameworks of a variety of macromolecular machines functioning in solution (14–16). 2-AP is a structural isomer of adenine that forms base pairs with thymine in DNA (and uridine in RNA), and the substitution of 2-AP for adenine in such bp does not significantly perturb the structure or stability of the resultant double helix. Furthermore, when these probes are used as dimer pairs, the CD spectrum primarily reflects the interaction of the transition dipoles of the two probes themselves and thus the local conformation of the DNA at those positions within the P/T DNA. The characteristic CD and fluorescence signals for 2-AP probes in nucleic acids occur at wavelengths of >300 nm, a spectral region in which the protein and the canonical nucleic acid components of the “macromolecular machines of gene expression” are otherwise transparent. In this study, we have examined the binding of Klenow and Klentaq polymerases to P/T DNA constructs that were designed to be comparable with the nucleic acid components of functioning replication complexes. By examining the low energy CD spectra of site-specifically placed 2-AP probes, we have been able to characterize base conformations at defined positions within the DNA to reveal conformational features of specific DNA bases bound at and near both the pol and the exo active sites of these polymerases. These measurements, in that they directly reflect the actual conformations of the DNA chains bound within the active sites of the functioning polymerase, have also provided a direct means to estimate the equilibrium distributions of primer ends between the two active sites for various P/T DNA constructs. 相似文献
14.
Properties of herpes simplex virus DNA polymerase and characterization of its associated exonuclease activity. 总被引:28,自引:0,他引:28
K W Knopf 《European journal of biochemistry》1979,98(1):231-244
Herpes simplex virus (HSV) DNA polymerase was isolated on a large-scale from African green monkey kidney cells infected with HSV type 1 (HSV-1) strain Angelotti. After DNA-cellulose chromatography the enzyme showed a specific activity of 48,000 units/mg protein. Three major single polypeptides with molecular weights of 144,000, 74,000 and 29,000 were copurified with the enzyme activity at the DNA-cellulose ste. By its chromatographic behavior and by template studies, the HSV DNA polymerase activity was clearly distinguishable from cellular alpha, beta and gamma DNA polymerase activities. Two exonucleolytic activities were found in the DNA-cellulose enzyme preparation. The main exonucleolytic activity, which degraded both single-stranded and double-stranded DNA to deoxynucleoside 5'-monophosphates, was separated by subsequent velocity sedimentation. The remaining exonucleolytic activity was not separable from the HSV DNA polymerase by several chromatographic steps and by velocity sedimentation at high ionic strength. This novel exonuclease and HSV DNA polymerase were equally sensitive both to phosphonoacetic acid and Zn2+ ions, inhibitors of the viral polymerase. Similar to the 3'-to-5'-exonuclease of procaryotic DNA polymerases and mammalian DNA polymerase delta, the HSV-polymerase-associated exonuclease catalyzed the removal of 3'-terminal nucleotides from the primer/template as well as the template-dependent conversion of deoxynucleoside triphosphates to monophosphates. 相似文献
15.
Effect of Deoxyribonucleic Acid Ligands on Deoxyribonucleases and Deoxyribonucleic Acid Polymerase I of Escherichia coli K-12 总被引:2,自引:1,他引:1
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Endonuclease I, exonuclease I, and exonuclease II-deoxyribonucleic acid (DNA) polymerase I activities are not vital functions in Escherichia coli, although the latter two enzymes have been indirectly shown to be involved in DNA repair processes. Acridines such as acridine orange and proflavine interfere with repair in vivo, and we find that such compounds inhibit the in vitro activity of exonuclease I and DNA polymerase I but stimulate endonuclease I activity and hydrolysis of p-nitrophenyl thymidine-5′-phosphate by exonuclease II. Another acridine, 10-methylacridinium chloride, binds strongly to DNA but is relatively inert both in vivo and in vitro. These experiments suggest that acridines affect enzyme activity by interacting with the enzyme directly as well as with DNA. Resulting conformational changes in the DNA-dependent enzymes might explain why similar acridines which form similar DNA complexes have such a wide range of physiological effects. Differential sensitivity of exonuclease I and DNA polymerase I to acridine inhibition relative to other DNA-dependent enzymes may contribute to the acridine sensitivity of DNA repair. 相似文献
16.
Characterization of the 5'' to 3'' exonuclease associated with Thermus aquaticus DNA polymerase. 总被引:8,自引:5,他引:3
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Thermus aquaticus DNA polymerase was shown to contain an associated 5' to 3' exonuclease activity. Both polymerase and exonuclease activities cosedimented with a molecular weight of 72,000 during sucrose gradient centrifugation. Using a novel in situ activity gel procedure to simultaneously detect these two activities, we observed both DNA polymerase and exonuclease in a single band following either nondenaturing or denaturing polyacrylamide gel electrophoresis: therefore, DNA polymerase and exonuclease activities reside in the same polypeptide. As determined by SDS-polyacrylamide gel electrophoresis this enzyme has an apparent molecular weight of 92,000. The exonuclease requires a divalent cation (MgCl2 or MnCl2), has a pH optimum of 9.0 and excises primarily deoxyribonucleoside 5'-monophosphate from double-stranded DNA. Neither heat denatured DNA nor the free oligonucleotide (24-mer) were efficient substrates for exonuclease activity. The rate of hydrolysis of a 5'-phosphorylated oligonucleotide (24-mer) annealed to M13mp2 DNA was about twofold faster than the same substrate containing a 5'-hydroxylated residue. Hydrolysis of a 5'-terminal residue from a nick was preferred threefold over the same 5'-end of duplex DNA. The 5' to 3' exonuclease activity appeared to function coordinately with the DNA polymerase to facilitate a nick translational DNA synthesis reaction. 相似文献
17.
INTERACTION OF POLY-L-LYSINE WITH CHROMATIN : Inhibition of In Situ RNA Synthesis Mediated by Escherichia coli RNA Polymerase
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RNA polymerase from Escherichia coli was used in conjunction with labeled nucleosides as an autoradiographic reagent to study the availability of template in the chromatin of fixed nuclei and chromosomes Sequential treatments of the tissues with acid and poly-L-lysine were used to compare the effect of these treatments on the availability of template with the previously reported effects on the in situ priming for Escherichia coli DNA polymerase Acid treatment was found to increase the in situ activity of both enzymes, while poly-L-lysine strongly inhibited the in situ reactions mediated by RNA and DNA polymerases. When the DNA polymerase reaction was previously carried out on alcohol-fixed chicken blood smears, leukocyte nuclei primed extensively for DNA synthesis. In contrast, we did not detect incorporation into intact nuclei of any cell type in alcohol-fixed blood smears that were treated with RNA polymerase. 相似文献
18.
Tomas T. Richardson Xiaohua Wu Brian J. Keith Pauline Heslop Anita C. Jones Bernard A. Connolly 《Nucleic acids research》2013,41(4):2466-2478
Archaeal family-B DNA polymerases bind tightly to deaminated bases and stall replication on encountering uracil in template strands, four bases ahead of the primer-template junction. Should the polymerase progress further towards the uracil, for example, to position uracil only two bases in front of the junction, 3′–5′ proof-reading exonuclease activity becomes stimulated, trimming the primer and re-setting uracil to the +4 position. Uracil sensing prevents copying of the deaminated base and permanent mutation in 50% of the progeny. This publication uses both steady-state and time-resolved 2-aminopurine fluorescence to show pronounced unwinding of primer-templates with Pyrococcus furiosus (Pfu) polymerase–DNA complexes containing uracil at +2; much less strand separation is seen with uracil at +4. DNA unwinding has long been recognized as necessary for proof-reading exonuclease activity. The roles of M247 and Y261, amino acids suggested by structural studies to play a role in primer-template unwinding, have been probed. M247 appears to be unimportant, but 2-aminopurine fluorescence measurements show that Y261 plays a role in primer-template strand separation. Y261 is also required for full exonuclease activity and contributes to the fidelity of the polymerase. 相似文献
19.
DNA polymerase from Escherichia coli (Pol I) and from avian myeloblastosis virus (AMV polymerase) were compared for the manner in which they catalyze the polymerization of deoxynucleotides upon a variety of synthetic and natural templates. It was found that the rates of nucleotide incorporation with different natural RNAs were similar. Both polymerases have an associated RNA endonuclease which hydrolyses RNA templates containing double-stranded regions. This activity depends on the presence of the complementary deoxynucleoside triphosphates, and/or polymerization. Both enzymes copy natural DNA, which has been sonicated and treated with E. coli exonuclease III, at the same rate. However, avian myeloblastosis virus DNA polymerase, which has no associated DNA exonuclease activity, is unable to copy double-stranded DNA and copies DNAase-treated DNA only 10% as well as Pol I. Pol I copied all the homopolymers investigated at a greater rate than AMV polymerase with the exception of poly(C) · oligo(dG). However, the initial rate of chain elongation, as measured by gel electrophoresis, was the same for the two polymerases, approximately 300 nucleotides incorporated per minute. Template saturation experiments show a stoichiometric relationship between template and enzyme at optimal rates of nucleotide incorporation which suggests that all enzyme molecules are potential catalysts. Enzyme saturation experiments indicate that not all enzyme molecules are “effectively” bound to a template. Fewer AMV polymerase than Pol I molecules are functionally bound to a particular template. From these data, it is concluded that the two polymerases elongate DNA chains in a similar way and that the manner in which the polymerases bind to a particular template accounts for the discrepancies found in their turnover numbers. 相似文献
20.
A key set of reactions for the initiation of new DNA strands during herpes
simplex virus-1 replication consists of the primase-catalyzed synthesis of
short RNA primers followed by polymerase-catalyzed DNA synthesis
(i.e. primase-coupled polymerase activity). Herpes primase
(UL5-UL52-UL8) synthesizes products from 2 to ∼13 nucleotides long.
However, the herpes polymerase (UL30 or UL30-UL42) only elongates those at
least 8 nucleotides long. Surprisingly, coupled activity was remarkably
inefficient, even considering only those primers at least 8 nucleotides long,
and herpes polymerase typically elongated <2% of the primase-synthesized
primers. Of those primers elongated, only 4–26% of the primers were
passed directly from the primase to the polymerase (UL30-UL42) without
dissociating into solution. Comparing RNA primer-templates and DNA
primer-templates of identical sequence showed that herpes polymerase greatly
preferred to elongate the DNA primer by 650–26,000-fold, thus accounting
for the extremely low efficiency with which herpes polymerase elongated
primase-synthesized primers. Curiously, one of the DNA polymerases of the host
cell, polymerase α (p70-p180 or p49-p58-p70-p180 complex), extended
herpes primase-synthesized RNA primers much more efficiently than the viral
polymerase, raising the possibility that the viral polymerase may not be the
only one involved in herpes DNA replication.Herpes simplex virus 1
(HSV-1)2 encodes seven
proteins essential for replicating its double-stranded DNA genome; five of
these encode the heterotrimeric helicase-primase (UL5-UL52-UL8 gene products)
and the heterodimeric polymerase (UL30-UL42 gene products)
(1,
2). The helicase-primase
unwinds the DNA at the replication fork and generates single-stranded DNA for
both leading and lagging strand synthesis. Primase synthesizes short RNA
primers on the lagging strand that the polymerase presumably elongates using
dNTPs (i.e. primase-coupled polymerase activity). These two protein
complexes are thought to replicate the viral genome on both the leading and
lagging strands (1,
2).Previous studies have focused on the helicase-primase and polymerase
separately. The helicase-primase contains three subunits, UL5, UL52, and UL8
in a 1:1:1 ratio
(3–5).
The UL5 subunit has helicase-like motifs and the UL52 subunit has primase-like
motifs, yet the minimal active complex that demonstrates either helicase or
primase activities contains both UL5 and UL52
(6,
7). Although the UL8 subunit
has no known catalytic activity, several functions have been proposed,
including enhancing helicase and primase activities, enhancing primer
synthesis on ICP8 (the HSV-1 single-stranded binding protein)-coated DNA
strands, and facilitating formation of the replisome
(8–12).
Although primase will synthesize short
(2–3
nucleotides long) primers on a variety of template sequences, synthesis of
longer primers up to 13 nucleotides long requires the template sequence,
3′-deoxyguanidine-pyrimidine-pyrimidine-5′
(13). Primase initiates
synthesis at the first pyrimidine via the polymerization of two purine NTPs
(13). Even after initiation at
this sequence, however, the vast majority of products are only 2–3
nucleotides long (13,
14).The herpes polymerase consists of the UL30 subunit, which has polymerase
and 3′ → 5′ exonuclease activities
(1,
2), and the UL42 subunit, which
serves as a processivity factor
(15–17).
Unlike most processivity factors that encircle the DNA, the UL42 protein binds
double-stranded DNA and thus directly tethers the polymerase to the DNA
(18). Using pre-existing DNA
primer-templates as the substrate, the heterodimeric polymerase (UL30-UL42)
incorporates dNTPs at a rate of 150 s–1, a rate much faster
than primer synthesis (for primers >7 nucleotides long, 0.0002–0.01
s–1) (19,
20).We examined primase-coupled polymerase activity by the herpes primase and
polymerase complexes. Although herpes primase synthesizes RNA primers
2–13 nucleotides long, the polymerase only effectively elongates those
at least 8 nucleotides long. Surprisingly, the polymerase elongated only a
small fraction of the primase-synthesized primers (<1–2%), likely
because of the polymerase elongating RNA primer-templates much less
efficiently than DNA primer-templates. In contrast, human DNA polymerase
α (pol α) elongated the herpes primase-synthesized primers very
efficiently. The biological significance of these data is discussed. 相似文献