Action of human endonucleases III and VIII upon DNA-containing tandem dihydrouracil

We have shown previously that endonuclease III from Escherichia coli, its yeast homolog Ntg1p and E. coli endonuclease VIII recognize single dihydrouracil (DHU) lesions efficiently. However, these enzymes have limited capacities for completely removing DHU, when the lesion is present on duplex DNA as a tandem lesion. A duplex 30-mer (duplex1920) containing tandem DHU lesions at positions 19 and 20 from the 5' terminus was used as a substrate for human endonuclease III (hNTH) and endonuclease VIII (NEIL1). Two cleavage products, 18beta and 19beta were formed, when duplex1920 was treated with hNTH. The 18beta corresponded to the expected beta-elimination product generated from duplex1920, when the 5'-DHU of the tandem DHU was processed by hNTH. Similarly, 19beta is the beta-elimination product generated, when the 3'-DHU of the tandem DHU was processed by hNTH; 19beta thus still contained a DHU lesion at the 3' terminus. When these hNTH reaction products were further treated with human APE1, a single new product that corresponded to an 18mer was observed. These data suggested that human APE1 can help to process the 3' terminals following the action of hNTH on DHU lesions. Similarly, when duplex1920 was treated with NEIL1, two cleavage products, 18p and 19p were observed. The 18p and 19p corresponded to the expected beta,delta-elimination products derived from NEIL1 induced cleavage at the 5'-DHU and 3'-DHU of the tandem DHU, respectively. The 3'-phosphoryl group present in 18p can be readily removed by T4 polynucleotide kinase (PNK) to yield an 18mer that is suitable for repair synthesis. However, 19p required the participation of both PNK and APE1 to generate the 18mer. Together, we suggest that the processing of DNA-containing tandem DHU lesions, initiated by hNTH and NEIL1 can be channeled into two sub-pathways, the PNK-independent, APE1-dependent and the PNK, APE1-dependent pathways, respectively.     

Multiply damaged sites in DNA: interactions with Escherichia coli endonucleases III and VIII.

Bursts of free radicals produced by ionization of water in close vicinity to DNA can produce clusters of opposed DNA lesions and these are termed multiply damaged sites (MDS). How MDS are processed by the Escherichia coli DNA glycosylases, endonuclease (endo) III and endo VIII, which recognize oxidized pyrimidines, is the subject of this study. Oligonucleotide substrates were constructed containing a site of pyrimidine damage or an abasic (AP) site in close proximity to a single nucleotide gap, which simulates a free radical-induced single-strand break. The gap was placed in the opposite strand 1, 3 or 6 nt 5' or 3' of the AP site or base lesion. Endos III and VIII were able to cleave an AP site in the MDS, no matter what the position of the opposed strand break, although cleavage at position one 5' or 3' was reduced compared with cleavage at positions three or six 5' or 3'. Neither endo III nor endo VIII was able to remove the base lesion when the gap was positioned 1 nt 5' or 3' in the opposite strand. Cleavage of the modified pyrimidine by endo III increased as the distance increased between the base lesion and the opposed strand break. With endo VIII, however, DNA breakage at the site of the base lesion was equivalent to or less when the gap was positioned 6 nt 3' of the lesion than when the gap was 3 nt 3' of the lesion. Gel mobility shift analysis of the binding of endo VIII to an oligonucleotide containing a reduced AP (rAP) site in close opposition to a single nucleotide gap correlated with cleavage of MDS substrates by endo VIII. If the strand break in the MDS was replaced by an oxidized purine, 7,8-dihydro-8-oxoguanine (8-oxoG), neither endo VIII cleavage nor binding were perturbed. These data show that processing of oxidized pyrimidines by endos III and VIII was strongly influenced by the position and type of lesion in the opposite strand, which could have a significant effect on the biological outcome of the MDS lesion.     

Excision of formamidopyrimidine lesions by endonucleases III and VIII is not a major DNA repair pathway in Escherichia coli

Proper maintenance of the genome is of great importance. Consequently, damaged nucleotides are repaired through redundant pathways. We considered whether the genome is protected from formamidopyrimidine nucleosides (Fapy•dA, Fapy•dG) via a pathway distinct from the Escherichia coli guanine oxidation system. The formamidopyrimidines are produced in significant quantities in DNA as a result of oxidative stress and are efficiently excised by formamidopyrimidine DNA glycosylase. Previous reports suggest that the formamidopyrimidine nucleosides are substrates for endonucleases III and VIII, enzymes that are typically associated with pyrimidine lesion repair in E.coli. We investigated the possibility that Endo III and/or Endo VIII play a role in formamidopyrimidine nucleoside repair by examining Fapy•dA and Fapy•dG excision opposite all four native 2′-deoxyribonucleotides. Endo VIII excises both lesions more efficiently than does Endo III, but the enzymes exhibit similar selectivity with respect to their action on duplexes containing the formamidopyrimidines opposite native deoxyribonucleotides. Fapy•dA is removed more rapidly than Fapy•dG, and duplexes containing purine nucleotides opposite the lesions are superior substrates compared with those containing formamidopyrimidine–pyrimidine base pairs. This dependence upon opposing nucleotide indicates that Endo III and Endo VIII do not serve as back up enzymes to formamidopyrimidine DNA glycosylase in the repair of formamidopyrimidines. When considered in conjunction with cellular studies [J. O. Blaisdell, Z. Hatahet and S. S. Wallace (1999) J. Bacteriol., 181, 6396–6402], these results also suggest that Endo III and Endo VIII do not protect E.coli against possible mutations attributable to formamidopyrimidine lesions.     

Enzymatic conversion of dihydrouracil into uracil

We found that some strains of Rhodotorula glutinis can oxideze dihydrourcil to uracil, and we converted dihydrouracil into uracil using the resting and immobilized cells of R. glutinis IFO-1389.The optimum pH of the conversion of dihydrouracil into uracil was 7.8. Oxygen supply was essential to the conversion. With resting cells, the addition of both o-phenanthroline and Triton X-100 caused increase of the yield of uracil about ten times as much as that with no addition.The conversion ratios of dihydrouracil into uracil using immobilized-cell beds, which were made with chitosan and glutaraldehyde, were 100, 98, and 77% when the concentration of dihydrouracil were 1, 2, and 3 (w/v)%, respectively, for 68 h at 30°C.     

Cleavage of phosphorothioate-substituted DNA by restriction endonucleases

M13 RF DNA was synthesized in vitro in the presence of various single deoxynucleoside 5'-O-(1-thiotriphosphate) phosphorothioate analogues, and the three other appropriate deoxynucleoside triphosphates using a M13 (+)-single-stranded template, Escherichia coli DNA polymerase I and T4 DNA ligase. The resulting DNAs contained various restriction endonuclease recognition sequences which had been modified at their cleavage points in the (-)-strand by phosphorothioate substitution. The behavior of the restriction enzymes AvaI, BamHI, EcoRI, HindIII, and SalI towards these substituted DNAs was investigated. EcoRI, BamHI, and HindIII were found to cleave appropriate phosphorothioate-substituted DNA at a reduced rate compared to normal M13 RF DNA, and by a two-step process in which all of the DNA is converted to an isolable intermediate nicked molecule containing a specific discontinuity at the respective recognition site presumably in the (+)-strand. By contrast, SalI cleaved substituted DNA effectively without the intermediacy of a nicked form. AvaI, however, is only capable of cleaving the unsubstituted (+)-strand in appropriately modified DNA.     

Synthesis and enzymatic processing of oligodeoxynucleotides containing tandem base damage.

Several studies have shown that ionizing radiation generates a wide spectrum of lesions to DNA including base modifications, abasic sites, strand breaks, crosslinks and tandem base damage. One example of tandem base damage induced by @OH radical inX-irradiated DNA oligomers is N -(2-deoxy-beta-d- erythro -pentofuranosyl)-formylamine/8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo). In order to investigate the biological significance of such a tandem lesion, both 8-oxo-7,8-dihydroguanine and formylamine were introduced into synthetic oligonucleotides at vicinal positions using the solid phase phosphoramidite method. For this purpose, a new convenient method of synthesis of 8-oxodGuo was developed. The purity and integrity of the modified synthetic DNA fragments were assessed using different complementary techniques including HPLC, polyacrylamide gel electrophoresis, electrospray and MALDI-TOF mass spectrometry. The piperidine test applied to the double modified base-containing oligonucleotides revealed the high alkaline lability of formylamine in DNA. In addition, various enzymatic experiments aimed at determining biochemical features of such multiply damaged sites were carried out using the synthetic substrates. The pro-cessing of the vicinal lesions by nuclease P1, snake venom phosphodiesterase, calf spleen phospho-diesterase and repair enzymes including Escherichia coli endonuclease (endo) III and Fapy-glycosylase was studied and is reported.     

Enzymatic processing of replication and recombination intermediates by the vaccinia virus DNA polymerase

Poxvirus DNA polymerases play a critical role in promoting virus recombination. To test if vaccinia polymerase (E9L) could mediate this effect by catalyzing the post-synaptic processing of recombinant joint molecules, we prepared substrates bearing a nick, a 3′-unpaired overhang, a 5′ overhang, or both 3′ and 5′ overhangs. The sequence of the 5′ overhang was also modified to permit or preclude branch migration across the joint site. These substrates were incubated with E9L, and the fate of the primer strand characterized under steady-state reaction conditions. E9L rapidly excises a mispaired 3′ strand from a DNA duplex, producing a meta-stable nicked molecule that is a substrate for ligase. The reaction was not greatly affected by adding an unpaired 5′ strand, but since such molecules cannot be processed into nicked intermediates, the 3′-ended strand continued to be subjected to exonucleolytic attack. Incorporating homology into the 5′ overhang prevented this and permitted some strand assimilation, but such substrates also promoted strand-displacement DNA synthesis of a type predicted by the 1981 Moyer and Graves model for poxvirus replication. Single-strand annealing reactions are used by poxviruses to produce recombinant viruses and these data show that virus DNA polymerases can process DNA in such a manner as to both generate single-stranded substrates for such reactions and to facilitate the final processing of the reaction products.     

Type III restriction endonucleases translocate DNA in a reaction driven by recognition site-specific ATP hydrolysis.

Type III restriction/modification systems recognize short non-palindromic sequences, only one strand of which can be methylated. Replication of type III-modified DNA produces completely unmethylated recognition sites which, according to classical mechanisms of restriction, should be signals for restriction. We have shown previously that suicidal restriction by the type III enzyme EcoP15I is prevented if all the unmodified sites are in the same orientation: restriction by EcoP15I requires a pair of unmethylated, inversely oriented recognition sites. We have now addressed the molecular mechanism of site orientation-specific DNA restriction. EcoP15I is demonstrated to possess an intrinsic ATPase activity, the potential driving force of DNA translocation. The ATPase activity is uniquely recognition site-specific, but EcoP15I-modified sites also support the reaction. EcoP15I DNA restriction patterns are shown to be predetermined by the enzyme-to-site ratio, in that site-saturating enzyme levels elicit cleavage exclusively between the closest pair of head-to-head oriented sites. DNA restriction is blocked by Lac repressor bound in the intervening sequence between the two EcoP15I sites. These results rule out DNA looping and strongly suggest that cleavage is triggered by the close proximity of two convergently tracking EcoP15I-DNA complexes.     

Oligodeoxynucleotides containing 7-deazaadenine: synthesis and recognition by restriction endonucleases

Deoxydecanucleotides containing a recognition sequence of Bg1 II and Sau 3AI, and their 7-deazaadenine analogs were synthesized by the phosphotriester method. The decanucleotides containing 7-deazaadenine in place of adenine were partially or strongly resistant to the hydrolysis by these restriction endonucleases.     

The interaction of DNA duplexes containing 2-aminopurine with restriction endonucleases EcoRII and SsoII.

Oligonucleotides containing 2-aminopurine (2-AP) in place of G or A in the recognition site of EcoRII (CCT/AGG) or SsoII (CCNGG) restriction endonucleases have been synthesized in order to investigate the specific interaction of DNA with these enzymes. Physicochemical properties (CD spectra and melting behaviour) have shown that DNA duplexes containing 2-aminopurine exist largely in a stable B-like form. 2-Aminopurine base paired with cytidine, however, essentially influences the helix structure. The presence of a 2-AP-C mismatch strongly reduces the stability of the duplexes in comparison with the natural double strand, indicated by a biphasic melting behaviour. SsoII restriction endonuclease recognizes and cleaves the modified substrate with a 2-AP-T mismatch in the centre of the recognition site, but it does not cleave the duplexes containing 2-aminopurine in place of inner and outer G, or both. EcoRII restriction endonuclease does not cleave duplexes containing 2-aminopurine at all. The two-substrate mechanism of EcoRII-DNA interaction, however, allows hydrolysis of the duplex containing 2-aminopurine in place of adenine in the presence of the canonical substrate.     

The cleavage of Drosophila melanogaster DNA by restriction endonucleases

Drosophila melanogaster DNA, together with λ and E. coli DNAs as controls, was digested with three different restriction endonucleases: EcoR_I, Hind, and Hae. The size distributions of the segments were characterized by gel electrophoresis. More than 85% of the D. melanogaster DNA was found in a broad distribution of segment lengths consistent with random location of restriction sites. However, some DNA was spared and recovered in very long (≥20500bp) segments. These segments proved to be mostly simple sequence DNA. No complex spared segments could be found in Hind and Hae digests, while 50% of the spared EcoR_I segments had a complexity exceeding that of the E. coli DNA spared by this enzyme. These data do not support the hypothesis that chromomeres contain long regions of purely tandemly repeating sequences.     

Enzymatic collapse of artificial polymer composite material containing double-stranded DNA

Large amounts of DNA-enriched biomaterials, such as salmon milts and shellfish gonads, are discarded as industrial waste around the world. Therefore, the utilizations of DNA with the specific function are important for the biomaterial science and the curce technology. We could convert the discarded DNA to an enzymatic collapsible material by the addition of DNA to the artificial polymer material, such as nylon. Although these DNA-artificial polymer composite materials were stable in water, these materials indicated the collapsibility at the DNA-hydrolyzed enzyme, such as Micrococcal nuclease, condition. Additionally, these collapsibilities under enzyme condition were controlled by the number of imino groups in the components of the artificial polymer. Furthermore, these composite materials could create the fiber form with a highly ordered molecular orientation by the reaction at the liquid/liquid interface. The DNA-artificial polymer composite materials may have the potential utility as a novel bio-, medical-, and environmental materials with the enzymatic collapsibility and degradability.     

Relation between Escherichia coli endonucleases specific for apurinic sites in DNA and exonuclease III.

Contradictory data have recently been published from two different laboratories on the presence vs absence of an intrinsic endonucliolytic activity of E. coli exonuclease III at apurinic sites in double-stranded DNA. It is shown here that an endonuclease activity of this specificity co-chromatographs exactly with exonuclease III on phosphocellulose and Sephadex G-75 columns, indicating that the endonuclease and exonuclease activities are due to the same enzyme. In addition, another E. coli endonuclease specific for apurinic sites exists, which can be separated from exonuclease III by the same chromatographic procedures.     

Thermodynamics of DNA target site recognition by homing endonucleases

The thermodynamic profiles of target site recognition have been surveyed for homing endonucleases from various structural families. Similar to DNA-binding proteins that recognize shorter target sites, homing endonucleases display a narrow range of binding free energies and affinities, mediated by structural interactions that balance the magnitude of enthalpic and entropic forces. While the balance of ΔH and TΔS are not strongly correlated with the overall extent of DNA bending, unfavorable ΔH_binding is associated with unstacking of individual base steps in the target site. The effects of deleterious basepair substitutions in the optimal target sites of two LAGLIDADG homing endonucleases, and the subsequent effect of redesigning one of those endonucleases to accommodate that DNA sequence change, were also measured. The substitution of base-specific hydrogen bonds in a wild-type endonuclease/DNA complex with hydrophobic van der Waals contacts in a redesigned complex reduced the ability to discriminate between sites, due to nonspecific ΔS_binding.     

Cleavage and protection of locked nucleic acid-modified DNA by restriction endonucleases

Locked nucleic acid (LNA) is one of the most prominent nucleic acid analogues reported so far. We herein for the first time report cleavage by restriction endonuclease of LNA-modified DNA oligonucleotides. The experiments revealed that RsaI is an efficient enzyme capable of recognizing and cleaving LNA-modified DNA oligonucleotides. Furthermore, introduction of LNA nucleotides protects against cleavage by the restriction endonucleases PvuII, PstI, SacI, KpnI and EcoRI.     

Digestion of highly modified bacteriophage DNA by restriction endonucleases.

The ability of thirty Type II restriction endonucleases to cleave five different types of highly modified DNA has been examined. The DNA substrates were derived from relatively large bacteriophage genomes which contain all or most of the cytosine or thymine residues substituted at the 5-position. These substituents were a proton (PBS1 DNA), a hydroxymethyl group (SP01 DNA), a methyl group (XP12 DNA), a glucosylated hydroxymethyl group (T4 DNA), or a phosphoglucuronated, glucosylated 4,5-dihydroxypentyl group (SP15 DNA). Although PBS1 DNA and SP01 DNA were digested by most of the enzymes, they were cleaved much more slowly than was normal DNA by many of them. 5-Methylcytosine-rich XP12 DNA and the multiply modified T4 and SP15 DNAs were resistant to most of these endonucleases. The only enzyme that cleaved all five of these DNAs was TaqI, which fragmented them extensively.     

Structural basis for sequence-dependent DNA cleavage by nonspecific endonucleases

Nonspecific endonucleases hydrolyze DNA without sequence specificity but with sequence preference, however the structural basis for cleavage preference remains elusive. We show here that the nonspecific endonuclease ColE7 cleaves DNA with a preference for making nicks after (at 3′O-side) thymine bases but the periplasmic nuclease Vvn cleaves DNA more evenly with little sequence preference. The crystal structure of the ‘preferred complex’ of the nuclease domain of ColE7 bound to an 18 bp DNA with a thymine before the scissile phosphate had a more distorted DNA phosphate backbone than the backbones in the non-preferred complexes, so that the scissile phosphate was compositionally closer to the endonuclease active site resulting in more efficient DNA cleavage. On the other hand, in the crystal structure of Vvn in complex with a 16 bp DNA, the DNA phosphate backbone was similar and not distorted in comparison with that of a previously reported complex of Vvn with a different DNA sequence. Taken together these results suggest a general structural basis for the sequence-dependent DNA cleavage catalyzed by nonspecific endonucleases, indicating that nonspecific nucleases could induce DNA to deform to distinctive levels depending on the local sequence leading to different cleavage rates along the DNA chain.     

DNA containing the base analogue 2-aminoadenine: preparation, use as hybridization probes and cleavage by restriction endonucleases.

The base analogue 2-aminoadenine (2,6-diaminopurine, D) has been introduced at selected positions into synthetic oligodeoxyribonucleotides and DNA by the combined use of chemical and enzymatic methods. 2-aminoadenine substitution for adenine introduces changes in the minor groove of DNA and creates an additional hydrogen bond in the Watson-Crick base pair with thymine. Oligonucleotide hybridization probes containing 2-aminoadenine showed increased selectivity and hybridization strength during DNA-DNA hybridization to phage or genomic target DNA. Properties of the base analogue with respect to DNA modifying enzymes were examined. 2-aminoadenine was used to probe minor groove determinants during the treatment of DNA by 12 restriction endonucleases. Inhibition of cleavage was found for several restriction enzymes.