Pancreatic Deoxyribonuclease Research Articles

Preparation and biological characteristics of a bovine acellular tendon fiber material.

Acellular tendon matrix is an ideal substitute for constructing tissue engineering ligaments, but using detergents causes damage to collagen and fibrin during the process of decellularization. In this study, fresh tendons were lyophilized and separated into fresh tendon fiber (FTF) bundles, and then the cellular components in FTF were removed to prepare acellular tendon fiber (ATF) without adding chemical detergent. H&E staining and DAPI fluorescence microscopy showed no nucleus and DNA residue. Compared with FTFs, the DNA content of ATFs was significantly lower without the collagen content change before and after decellularization. The microstructure of collagen fibrils in ATFs was intact under scanning electron microscopy (SEM), and the maximum tensile load and elastic modulus between FTFs and ATFs were not statistically different. The ATF bundles were cultured with SD rat tenocytes for 72 hr and cells attachment to fiber surfaces were observed under SEM. ATF bundles were then implanted into paraspinal muscles, and histological analysis showed fibroblast-like cells within the ATFs and was similar to the control group (fresh tendon autograft) in morphology. H&E staining showed that the number of lymphocytes and plasma cells in ATF was less than that in fresh tendon autograft. ATF bundles were twisted into linear fiber materials by hand, of which the maximum breaking strength was similar to silk with same diameter. These findings demonstrated that ATFs retain their original fibril structure and mechanical properties after decellularization by trypsin and pancreatic deoxyribonuclease without detergent. Lyophilized ATFs linear fiber material provides the possibility of preparing personalized ligament and other tissue engineering scaffolds.

Benzimidazoles as novel deoxyribonuclease I inhibitors.

Inhibitory potential of 19 benzimidazoles against bovine pancreatic deoxyribonuclease I (DNase I) was investigated in vitro. Three compounds inhibited DNase I with IC50 below 100 μM and proved to be more potent DNase I inhibitors than crystal violet (IC50 = 351.82 ± 29.41 μM), used as a positive control. Compound 9 showed the most potent DNase I inhibition with an IC 50 value of 79.46 ± 11.75 μM. To further explore the relationship between inhibitory activity and 2D pharmacophore features, Pharma/E-State R-group quantitative structure-activity relationship (RQSAR) models were generated and validated using Schrödinger Suite. RQSAR models showed a significant enhancement of benzimidazoles activity using hydrogen-bond acceptor substituents at the R2, R3, and R4 positions, or aryl substituents at the R4 position. TheSite Finder module and molecular docking defined the benzimidazoles interactions with the most important catalytic residues of DNase I, including H-acceptor interaction with residue His 134 and His 252 and/or H-donor interaction with residue Glu 39. We also found a positive correlation between IC50 inhibition values and relative binding free energies of the most active benzimidazoles. In addition, a molecular dynamics simulation was performed for DNase I-compound 9 docking complex in Desmond. Trajectory analysis showed that docking complex and intermolecular interactions werestable throughout the entire production part of simulations. Furthermore, the results of protein structure alignment module suggested the potential translational impact of benzimidazoles against human DNase I. Due to the significant involvement of DNase I in the pathophysiology of many disease conditions, benzimidazoles as DNase I inhibitors could have potential therapeutic applications.

Overexpression and Characterization of Bovine Pancreatic Deoxyribonuclease I in Saccharomyces cerevisiae and Pichia pastoris

In the present study, we investigated the overexpression and characterization of bovine pancreatic (bp)- DNase I in Saccharomyces cerevisiae and Pichia pastoris. The bp-DNase I gene was fused in frame with the GAL10 promoter, , and GAL7 terminator sequences, resulting in the plasmid, pGAL--DNaseI (6.4 kb). Also, the bp-DNase I gene was fused in frame with the AOX1 promoter, , and AOX1 terminator sequences, resulting in the plasmid, pPEXI (8.8 kb). The recombinant plasmids, pGAL--DNaseI and pPEXI were introduced into S. cerevisiae and P. pastoris host cells, respectively. When the transformed yeast cells were cultured at for 48 h in galactose or methanol medium, bp-DNase I was overexpressed and the most of activity was found in the extracellular fraction. P. pastoris transformant activity showed 45.5 unit/mL in the culture medium at 48 h cultivation, whereas S. cerevisiae transformant revealed 37.7 unit/mL in the extracellular fraction at 48 h cultivation. The enzymatic characteristics, such as DNA cleavage and half life were investigated. Treatment of the recombinant DNase I from P. pastoris induced degradation of the calf thymus DNA within 1 minute, and this DNA degradation rate was higher than that of commercial bp-DNase I (SIGMA) and the recombinant DNase I from S. cerevisiae.

Extraction and purification of bovine pancreatic Deoxyribonuclease I

Samples of bovine pancreatic and calf thymus glands were collected from local slaughterers to extract deoxyribonuclease I enzyme (DNase I) and DNA respectively. Crude extract was prepared by using cooled distilled water and 0.25 N sulfuric acid .This study show that the best condition for the crude extract activity was 17 U/ml and 1 hr. Incubated period reaction with its substrate.The bovine pancreatic DNase I was purified by several steps of precipitation using ammonium sulphate to 529.4 times, with an enzymes yield 6.35%. Enzyme characterization studies indicate that: it is active at 2.7 U/ml, and 50 µg /ml DNA after 30 min of reaction time, the enzyme activity was higher at pH 6.5 and it showed stability at pH 9. The maximum enzyme activity was reported at 50 °C The results obtained from the role of metal ions (Mg+2, Mn+2) on enzyme activity indicate that these ions stimulated the enzyme activity while the Ca+2 and Cu+2 had lower stimulating activity on the enzyme but Ag+1 and Hg+2 showed inhibitory effect on enzyme activity. In addition the enzyme activity was inhibited by using denaturing Sodium dodecyl sulphate (SDS), chelating agents Ethylene diamine tetra acetic acid (EDTA), and reducing agents (2-Merceptoethanol and Urea) , and maintained it's activity when incubated with Phenyl Methyl Sulphonyl Floride (PMSF) .

Putative Secondary Active Site of Bovine Pancreatic Deoxyribonuclease I

Previous structural studies based on the co-crystal of a complex between bovine pancreatic deoxyribonuclease I (bpDNase I) and a double-stranded DNA octamer d(GCGATCGC)(2) have suggested the presence of a putative secondary active site near Ser43. In our present study, several crucial amino acid residues postulated in this putative secondary active site, including Thr14, Ser43, and His44 were selected for site-directed mutagenesis. A series of single, double and triple mutants were thus constructed and tested for their DNase I activity by hyperchromicity assay. Substitution of each or both of Thr14 and Ser43 by alanine results in mutant enzymes retaining 30-70% of WT bpDNase I activity. However, when His44 was replaced by aspartic acid, either in the single, double, or triple mutant, the enzyme activities were drastically decreased to 0.5-5% that of WT bpDNase I. Interestingly, when cysteine was substituted for Thr14 or Ser43, the specific DNase activities of the mutant enzymes were substantially increased by 1.5-100-fold, comparing to their alanine substitution mutant counterparts. Two other more sensitive DNase activity assay method, plasmid scission and zymogram analyses further confirm these observations. These results suggested that His44 may play a critical role in substrate DNA binding in this putative secondary active site, and introduction of sulfhydryl groups at Thr14 and Ser43 may facilitate Mn(2+)-coordination and further contribute to the catalytic activity of bpDNase I.

2-Nitro-5-Thiosulfobenzoic Acid as a Novel Inhibitor Specific for Deoxyribonuclease I

Bovine pancreatic deoxyribonuclease I (bpDNase I) contains four cysteine residues forming two disulfide bonds. Though there are no free sulfhydryl groups, incubation of bpDNase I with 2-nitro-5-thiosulfobenzoic acid (NTSB) in the presence of Ca(2+) or Mg(2+) at pH 7.5 results in inactivation of the enzyme. Amino acid analysis shows that NTSB-treated bpDNase I still contains all 4 half-cystine residues. The only amino acid residues having reduced values are threonine and serine, indicating that these may be the reaction sites for NTSB. Plasmid scission assay and circular dichroism analysis reveal the structural integrity of the inactivated enzyme. Treatment of bpDNase I with NTSB does not result in fragmentation, as demonstrated by SDS-PAGE analysis. NTSB binds bpDNase I through covalent modification, since dialysis and gel filtration can not reverse the inactivation reaction. However, after dilution into an acid buffer of pH 4.7, the inactivated enzyme regains about 40% of its initial activity, suggesting a reversible inactivation by acid treatment. NTSB does not inactivate DNase II, ribonuclease, chymotrypsin and lysozyme, while it effectively inactivates rat parotid DNase I. These results strongly suggest that NTSB can be considered as a novel inhibitor specific for DNase I.

Construction and characterization of a bifunctional enzyme with deoxyribonuclease I and thioredoxin-like activities

One large essential (C173–C209) and one small nonessential (C101–C104) disulfide loops occur in bovine pancreatic deoxyribonuclease I (bpDNase I). In our recent study, the reduced nonessential disulfide (-CESC-), which is structurally homologous to the active-site motif (-CGPC-) of thioredoxin, was shown to have thioredoxin-like activity. In order to gain further insight into the potential redox activity of the nonessential disulfide in bpDNase I, four double (GP, PG, WK, and KW) and two quadruple (WGPK, KPGW) mutants were constructed. Most of the mutant enzymes possess similar specific DNase activities as that of WT bpDNase I, while KPGW exhibited only half of the activity, possibly due to gross structural alteration, as revealed by CD analysis. All these mutants were able to accelerate the rate of insulin precipitation. The highest thioredoxin-like activity (66%) measured for WGPK indicated that the conserved sequence (-WCGPCK-) of thioredoxin is crucial for its redox activity. Our results suggested that engineering of the nonessential disulfide in bpDNase I was able to generate a novel bifunctional enzyme with enhanced disulfide/dithiol exchange reactivity, while retaining its full DNA-hydrolyzing activity.

Probing the catalytic mechanism of bovine pancreatic deoxyribonuclease I by chemical rescue

Previous structural and mutational studies of bovine pancreatic deoxyribonuclease I (bpDNase I) have demonstrated that the active site His134 and His252 played critical roles in catalysis. In our present study, mutations of these two His residues to Gln, Ala or Gly reduced the DNase activity by a factor of four to five orders of magnitude. When imidazole or primary amines were added exogenously to the Ala or Gly mutants, the residual DNase activities were substantially increased by 60–120-fold. The rescue with imidazole was pH- and concentration-dependent. The pH-activity profiles showed nearly bell-shaped curves, with the maximum activity enhancement for H134A at pH 6.0 and that for H252A at pH 7.5. These findings indicated that the protonated form of imidazole was responsible for the rescue in H134A, and the unprotonated form was for that in H252A, prompting us to assign unambiguously the roles for His134 as a general acid, and His252 as a general base, in bpDNase I catalysis.

Structure and Function of Bovine Pancreatic Deoxyribonuclease I

Bovine pancreatic deoxyribonuclease I (bpDNase), the first DNase discovered, is the best characterized among various types of DNase. A catalytic mechanism has been suggested based on the X-ray structure of the bpDNase-octamer complex. In this review, we will focus on three aspects: 1) the distinctive functions of the two structural calcium atoms; 2) the biological functions of the two disulfides; and 3) the involvement of the N- and C-terminal fragments in the enzyme folding for activity.

Antiprotease Function of Airway Secretions in Purulent Tracheobronchitis

Unopposed activity of the serine protease, human leukocyte elastase (HLE), is detectable in the airways of patients with purulent tracheobronchitis. The aim of this study was to assess the compartmentalization of HLE activity in the liquid sol phase and the solid gel phase of airway secretions. Seventy samples of tracheobrochial aspirates were obtained from patients who had hypersecretion and were receiving mechanical ventilation. Samples were separated into sol and gel ("mucous pellet") phases, and HLE activity was measured using chromogenic substrate degradation. HLE was eluted from the mucous pellet using hypertonic saline solution, 1 mol/L, or bovine pancreatic deoxyribonuclease (DNase), 16 micromol/L. HLE activity partitioned between the sol and gel phases of the secretions, with most of the activity present in the gel phase (32:1 ratio of gel to sol HLE activity). The activity of HLE was 95% inhibited when bound to the gel phase, but activity appeared to be largely restored after elution from the gel phase. The gel phase was capable of binding additional exogenous HLE, and its binding capacity for exogenous HLE was not saturated by concentrations that exceeded the highest clinically relevant HLE levels (1.1 mg/mL). Hypertonic saline solution and DNase I efficiently liberated endogenous and exogenous gel phase-bound HLE activity, suggesting that electrostatic bonds and DNA, respectively, play important roles in binding HLE to the gel phase. The solid phase of airway secretions is a more important modulator of elastase-antielastase balance than has been previously recognized.

Molecular evolution of shark and other vertebrate DNases I

We purified pancreatic deoxyribonuclease I (DNase I) from the shark Heterodontus japonicus using three-step column chromatography. Although its enzymatic properties resembled those of other vertebrate DNases I, shark DNase I was unique in being a basic protein. Full-length cDNAs encoding the DNases I of two shark species, H. japonicus and Triakis scyllia, were constructed from their total pancreatic RNAs using RACE. Nucleotide sequence analyses revealed two structural alterations unique to shark enzymes: substitution of two Cys residues at positions 101 and 104 (which are well conserved in all other vertebrate DNases I) and insertion of an additional Thr or Asn residue into an essential Ca(2+)-binding site. Site-directed mutagenesis of shark DNase I indicated that both of these alterations reduced the stability of the enzyme. When the signal sequence region of human DNase I (which has a high alpha-helical structure content) was replaced with its amphibian, fish and shark counterparts (which have low alpha-helical structure contents), the activity expressed by the chimeric mutant constructs in transfected mammalian cells was approximately half that of the wild-type enzyme. In contrast, substitution of the human signal sequence region into the amphibian, fish and shark enzymes produced higher activity compared with the wild-types. The vertebrate DNase I family may have acquired high stability and effective expression of the enzyme protein through structural alterations in both the mature protein and its signal sequence regions during molecular evolution.

Involvement of the N- and C-terminal fragments of bovine pancreatic deoxyribonuclease in active protein folding.

The three-dimensional structure of bovine pancreatic (bp) DNase revealed that its N- and C-termini form an antiparallel beta-sheet structure. The involvement of this beta-sheet structure in the active protein folding of bpDNase was thus investigated via a series of deletion and substitution variants. Several substitution variants of N-terminal Leu1 and C-terminal Leu259, and one variant with only the last Thr260 deleted, remained fully active. However, the other deletion variants, in which 2-10 amino acid residues were removed from the C- or N-terminus, all lost the DNase activity. The results indicated that the backbone hydrogen bonding in the antiparallel beta-sheet, rather than the side-chain interactions, is crucial for the correct protein folding. When the deletion variants were complemented with synthetic peptides of the deleted N- or C-terminal sequences, the DNase activity was generated. The highest DNase activity was generated when the C-terminal 10-residue-deleted brDNase(Delta251-260) was admixed with the C-terminal 10-residue peptide (peptide C10) in a molar ratio of 1:400. The noncovalent binding between brDNase(Delta251-260) and peptide C10 exhibited a dissociation constant of 48 microM. Circular dichroism spectra showed that the deletion variants were partially folded with mainly helical structures and that admixture with corresponding peptides facilitated their folding into the nativelike beta-sheet-rich structure. Thermal denaturation profiles also revealed that the transition temperature for brDNase(Delta251-260) was increased from 55 to 63 degrees C after incubation with peptide C10. The folding activation process for the deletion variant occurred in two stages, and Ca(2+) was required.

Biological functions of the disulfides in bovine pancreatic deoxyribonuclease.

We characterized the biochemical functions of the small nonessential (C101-C104) and the large essential (C173-C209) disulfides in bovine pancreatic (bp) DNase using alanine mutants [brDNase(C101A)] and [brDNase(C173A) and brDNase(C209A)], respectively. We also characterized the effects of an additional third disulfide [brDNase(F192C/A217C)]. Without the Ca(2+) protection, bpDNase and brDNase(C101A) were readily inactivated by trypsin, whereas brDNase(F192C/A217C) remained active. With Ca(2+), all forms of DNase, except for brDNase(C101A), were protected against trypsin. All forms of DNase, after being dissolved in 6 M guanidine-HCl, were fully reactivated by diluting into a Ca(2+)-containing buffer. However, when diluted into a Ca(2+)-free buffer, bpDNase and brDNase(C101A) remained inactive, but 60% of the bpDNase activity was restored with brDNase(F192C/A217C). When heated, bpDNase was inactivated at a transition temperature of 65 degrees C, brDNase(C101A) at 60 degrees C, and brDNase(F192C/A217C) at 73 degrees C, indicating that the small disulfide, albeit not essential for activity, is important for the structural integrity, and that the introduction of a third disulfide can further stabilize the enzyme. When pellets of brDNase(C173A) and brDNase(C209A) in inclusion bodies were dissolved in 6 M guanidine-HCl and then diluted into a Ca(2+)-containing buffer, 10%-18% of the bpDNase activity was restored, suggesting that the "essential" disulfide is not absolutely crucial for enzymatic catalysis. Owing to the structure-based sequence alignment revealing homology between the "nonessential" disulfide of bpDNase and the active-site motif of thioredoxin, we measured 39% of the thioredoxin-like activity for bpDNase based on the rate of insulin precipitation (DeltaA650nm/min). Thus, the disulfides in bpDNase not only play the role of stabilizing the protein molecule but also may engage in biological functions such as the disulfide/dithiol exchange reaction.

A single amino acid substitution of Leu130Ile in snake DNases I contributes to the acquisition of thermal stability.

We purified pancreatic deoxyribonucleases I (DNases I) from three snakes, Elaphe quadrivirgata, Elaphe climacophora and Agkistrodon blomhoffii, and cloned their cDNAs. Each mature snake DNase I protein comprised 262 amino acids. Wild-type snake DNases I with Leu130 were more thermally unstable than wild-type mammalian and avian DNases I with Ile130. After substitution of Leu130Ile, the thermal stabilities of the snake enzymes were higher than those of their wild-type counterparts and similar to mammalian wild-type enzyme levels. Conversely, substituting Ile130Leu of mammalian DNases I made them more thermally unstable than their wild-type counterparts. Therefore, a single amino acid substitution, Leu130Ile, might be involved in an evolutionally critical change in the thermal stabilities of vertebrate DNases I. Amphibian DNases I have a Ser205 insertion in a Ca2+-binding site of mammalian and avian enzymes that reduces their thermal stabilities [Takeshita, H., Yasuda, T., Iida, R., Nakajima, T., Mori, S., Mogi, K., Kaneko, Y. & Kishi, K. (2001) Biochem. J.357, 473-480]. Thus, it is plausible that the thermally stable wild-type DNases I of the higher vertebrates, such as mammals and birds, have been generated by a single Leu130Ile substitution of reptilian enzymes through molecular evolution following Ser205 deletion from amphibian enzymes. This mechanism may reflect one of the evolutionary changes from cold-blooded to warm-blooded vertebrates.

The distinctive functions of the two structural calcium atoms in bovine pancreatic deoxyribonuclease.

The two amino acid residues, Asp 99 and Asp 201, involved in the coordination of the two calcium atoms in the X-ray structure of bovine pancreatic (bp) DNase, were individually changed by site-directed mutagenesis. The two altered proteins, brDNase(D99A) and brDNase(D201A) were expressed in Escherichia coli and purified by anion exchange chromatography. Equilibrium dialysis showed that mutation destroyed one Ca(2+)-binding site each in brDNase(D99A) and brDNase(D201A). Compared with bpDNase, the Vmax value for brDNase(D99A) remained unchanged and that for brDNase(D201A) was decreased, whereas the K(m) values for the two variants were increased two- to threefold when the DNA hydrolytic hyperchromicity assay was used. Like bpDNase, brDNase(D99A) was able to make double scission on duplex DNA with Mg(2+) plus Ca(2+) and was effectively protected by Ca(2+) from the trypsin inactivation. But under the same conditions, brDNase(D201A) lost the double-scission ability and was not protected by Ca(2+). Nevertheless, the two variant proteins retained the characteristics of the Ca(2+)-induced conformational changes and the Ca(2+) protection against the beta-mercaptoethanol disruption of the essential disulfide bond, suggesting that other weaker Ca(2+)-binding sites not found in the X-ray structure were responsible for these properties. Therefore, the two structural calcium atoms are not for maintaining the overall conformation of the active DNase, as it has been indicated in the X-ray analysis, but rather play the role in the fine-tuning of the DNase activity.

Molecular, biochemical and immunological studies of hen pancreatic deoxyribonuclease I

Deoxyribonuclease I (DNase I) was purified from the hen pancreas to electrophoretic homogeneity using six-step column chromatography. The purified enzyme showed a molecular mass of about 33 kDa and maximum activity at pH 7.0. It required divalent cations, Mg 2+ and Ca 2+, for its activity and was inhibited by EDTA, EGTA and an antibody specific to the purified enzyme but not by G-actin. A 1066-bp cDNA encoding hen DNase I was constructed from the total RNA of a hen pancreas using a combination of the reverse transcriptase–polymerase chain reaction and rapid amplification of cDNA ends methods, followed by sequencing. The cDNA was expressed in Escherichia coli, and the recombinant polypeptide exhibited significant enzyme activity. The mature hen DNase I protein was found to consist of 262 amino acids. In human and bovine DNase I four amino acid residues, Glu-13, Tyr-65, Val-67 and Ala-114 are involved in actin binding, whereas in the hen DNase I these positions were occupied by Asp, Phe, Ser and Phe, respectively. A survey of the DNase I distribution in 15 hen tissues showed that the pancreas had the highest levels of both DNase I enzyme activity and DNase I gene expression. The results of our phylogenetic and immunological analyses indicate that the hen DNase I is not closely related to the mammalian enzymes. This is the first report in which has been described the results of molecular, biochemical and immunological analyses on hen DNase I.

Effect of 17β estradiol and progesterone on the conformation of the chromatin of the liver of female Japanese quail during aging

Plasma levels of 17β estradiol and progesterone were measured by radioimmunoassay in immature, adult and old female Japanese quails. The levels of progesterone and the progesterone/estradiol ratio were maximum in adult, egg laying birds. Conformation of the chromatin of the liver of birds of various ages before and after administration of steroid hormones was studied by digesting the nuclei with micrococcal nuclease (MNase) and pancreatic deoxyribonuclease I (DNase I) followed by electrophoretic resolution of the DNA fragments. The pattern of bands shows that the chromatin of the adult is more sensitive to DNase I and MNase than that of young and old birds. Administration of 17β estradiol and progesterone enhances the digestion of the chromatin by DNase I and MNase in old birds. Such enhancement does not occur in adult birds as the chromatin is already in relaxed and open conformation due to the high levels of the hormones already present at this age.

Site-directed mutagenesis of phosphate-contacting amino acids of bovine pancreatic deoxyribonuclease I.

Bovine pancreatic deoxyribonuclease I (DNase I) is an endonuclease which cleaves double-stranded DNA. Cocrystal structures of DNase I with oligonucleotides have revealed interactions between the side chains of several amino acids (N74, R111, N170, S206, T207, and Y211) and the DNA phosphates. The effects these interactions have on enzyme catalysis and DNA hydrolysis selectivity have been investigated by site-directed mutagenesis. Mutations to R111, N170, T207, and Y211 severely compromised activity toward both DNA and a small chromophoric substrate. A hydrogen bond between R111 (which interacts with the phosphate immediately 5' to the cutting site) and the essential amino acid H134 is probably required to maintain this histidine in the correct orientation for efficient hydrolysis. Both T207 and Y211 bind to the phosphate immediately 3' to the cleavage site. Additionally, T207 is involved in binding an essential, structural, calcium ion, and Y211 is the nearest neighbor to D212, a critical catalytic residue. N170 interacts with the scissile phosphate and appears to play a direct role in the catalytic mechanism. The mutation N74D, which interacts with a phosphate twice removed from the scissile group, strongly reduced DNA hydrolysis. However, a comparison of DNase I variants from several species suggests that certain amino acids, which allow interaction with phosphates (positively charged or hydrogen bonding), are tolerated. S206, which binds to a DNA phosphate two positions away from the cleavage site, appears to play a relatively unimportant role. None of the enzyme variants, including a triple mutation in which N74, R111, and Y211 were altered, affected DNA hydrolysis selectivity. This suggests that phosphate binding residues play no role in the selection of DNA substrates.

Conversion of bovine pancreatic DNase I to a repair endonuclease with a high selectivity for abasic sites.

Bovine pancreatic deoxyribonuclease I (DNase I) is a nuclease of relatively low specificity which interacts with DNA in the minor groove. No contacts are made between the protein and the major groove of the nucleic acid. DNase I is structurally homologous to exonuclease III, a DNA-repair enzyme with multiple activities. One of the main differences between the two enzymes is the presence of an additional alpha-helix in exonuclease III, in a position suggestive of interaction with the major groove of DNA. Recombinant DNA techniques have been used to add 14 amino acids, comprising the 10 amino acids of the exonuclease III alpha-helix flanked by a glycine rich region, to DNase I. The polypeptide has been inserted after serine 174, an amino acid on the surface of DNase I corresponding to the location of the extra alpha-helix in exonuclease III. The recombinant protein, DNase-exohelix, has been purified and its catalytic activities towards DNA investigated. The recombinant protein demonstrated a high selectivity for endonucleolytic cleavage at abasic sites in DNA, a property of exonuclease III but not native DNase I. Thus the insertion of 14 amino acids at Ser174, converts DNase I to an exonuclease III-like enzyme with DNA-repair properties.

A single N-2-acetylaminofluorene adduct alters the footprint of T7 (exo−) DNA polymerase bound to a model primer–template junction

Bovine pancreatic deoxyribonuclease I (DNaseI) has been used to footprint T7 (exo−) DNA polymerase bound to a model primer–template junction. The polymerase was blocked at a specific position either by the omission of dCTP from the reaction mix or by the presence of a N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dGuo-AAF) adduct. This lesion has been shown to be a severe block for several DNA polymerases, both in in vitro primer elongation experiments, and during the in vivo replication of AAF-monomodified single-stranded vectors. The footprints obtained with unmodified primer–template DNA define two protected domains separated by an inter-region that remains sensitive to DNaseI, and several hypersensitive sites located on both strands. Binding of the polymerase to AAF monomodified duplexes results in the same protection pattern as that obtained with the unmodified duplexes. However, the hypersensitive sites either disappear or are dramatically reduced. The results suggest that the AAF lesion alters the correct positioning of the duplex DNA within the polymerase cleft.