Localization of canonical single-strand breaks in DNA of the Pseudomonas aeruginosa bacteriophage phi kF77

Bacteriophages phi k of P. aeruginosa were characterized by the presence of T4 DNA-ligase-repaired, single-chain breaks in their genome. A restriction map was constructed for one of these phages (phi kF77) with restriction endonucleases SalI, HindIII, EcoRI, MluI, XbaI and ClaI. phi kF77 DNA was resistant to the cleavage by BamHI, BglII, HpaI, PstI, PvuII and XhoI endonucleases. Single-chain breaks were mapped by means of electron microscopy of partially denatured DNA molecules, electrophoretic studies of denatured DNA and S1-analysis. Four major nicks were thus located which were revealed in 33 to 83% of DNA molecules. On the basis of mutual hybridization of single-strand DNA fragments it was shown that all nicks are located in one of the phi kF77 DNA chains. S1-treated hybrids of 32P-labeled single-strand fragments with intact DNA chain were used for DNA orientation. The physical map of phi kF77 DNA was constructed.     

Nucleotide sequence at the site of single-strand DNA breaks in Pseudomonas aeruginosa bacteriophage phi kF77

It is known that DNA molecules from the phage group phi k specific to Pseudomonas aeruginosa possess single-strand breaks (nicks). The sequences around the nicks in the bacteriophage phi kF77 DNA have been determined by various methods. In addition, an EcoRV-HindIII fragment, containing a nick, was cloned into the plasmid pUC9 and sequenced by Maxam-Gilbert technique. The sequence common for all nicks was CCTAohpCTCCGG.     

Rapid inactivation of bacteriophage T7 by ascorbic acid is repairable

Treatment of bacteriophage T7 with ascorbic acid resulted in the rapid accumulation of single-strand breaks in the DNA with double-strand breaks appearing only after incubation times of 20 min or longer. The single-strand breaks were responsible for a rapid inactivation of the phage as assayed by immediate plating of the phage-bacteria mixture on nutrient agar. Incubation of the phage-bacteria mixture in liquid medium prior to plating allowed a host cell reactivation process to repair the nicks and reactivate the phage. Non-reversible inactivation of the phage was a slower process which could be correlated with the appearance of double-strand breaks in the phage DNA. Host cell reactivation of the phage was also manifested in the phenomena of delayed lysis and delayed appearance of the concatemeric DNA replication intermediate.     

Genomic Analysis of Pseudomonas putida Phage tf with Localized Single-Strand DNA Interruptions

The complete sequence of the 46,267 bp genome of the lytic bacteriophage tf specific to Pseudomonas putida PpG1 has been determined. The phage genome has two sets of convergently transcribed genes and 186 bp long direct terminal repeats. The overall genomic architecture of the tf phage is similar to that of the previously described Pseudomonas aeruginosa phages PaP3, LUZ24 and phiMR299-2, and 39 out of the 72 products of predicted tf open reading frames have orthologs in these phages. Accordingly, tf was classified as belonging to the LUZ24-like bacteriophage group. However, taking into account very low homology levels between tf DNA and that of the other phages, tf should be considered as an evolutionary divergent member of the group. Two distinguishing features not reported for other members of the group were found in the tf genome. Firstly, a unique end structure – a blunt right end and a 4-nucleotide 3′-protruding left end – was observed. Secondly, 14 single-chain interruptions (nicks) were found in the top strand of the tf DNA. All nicks were mapped within a consensus sequence 5′-TACT/RTGMC-3′. Two nicks were analyzed in detail and were shown to be present in more than 90% of the phage population. Although localized nicks were previously found only in the DNA of T5-like and phiKMV-like phages, it seems increasingly likely that this enigmatic structural feature is common to various other bacteriophages.     

Role of genes 46 and 47 in bacteriophage T4 reproduction. II. Formation of gaps on parental DNA of polynucleotide ligase defective mutants

The nature of nucleolytic activity regulated by genes 46 and 47 of bacteriophage T4 was studied by examining the metabolism of parental DNA of phages carrying a mutation in polynucleotide ligase gene (lig) and an additional mutation in one of the following D0 genes (D0 genes are necessary for T4 DNA synthesis): 32, 43 (DNA polymerase  pol), 44 and 45. Polynucleotide ligase and DNA polymerase were used to distinguish nicks (phosphodiester bond interruptions on duplex DNA) from gaps (interruptions with missing nucleotides). In non-permissive hosts, parental DNA of double mutants (lig, D0) accumulated both single- and double-strand breaks. Up to 30% of this DNA eventually became acid-soluble. An additional mutation in gene 46 (or 47) did not prevent accumulation of double- and single-strand breaks but did prevent degradation to the acid-soluble state. The majority of the single-strand breaks on (lig, D0)-DNA were presumed to be gaps since, after extraction from infected host cells, they were repaired by ligase plus DNA polymerase but not by ligase alone. In contrast, the majority of the single-strand breaks on parental DNA of (lig, D0, 46) or (lig, pol, 47) were repaired by ligase alone, suggesting nicks, rather than gaps. These observations suggest that (i) genes 46 and 47 regulate, either directly or indirectly, an exonuelease activity which can attack T4 DNA at nicks to create gaps, and (ii) T4 DNA polymerase, and the products of genes 32, 44 and 45 are necessary to prevent nicks from becoming gaps in vivo. Possible roles for genes 46 and 47 in T4 DNA replication and in recombination are discussed.     

Co-fractionation of an endonuclease activity during the purification of DNA polymerase-alpha from regenerating rat liver. Properties and separation from DNA polymerase.

The presence of endonuclease activity associated with DNA polymerase was detected during the purification of high-molecular-weight DNA polymerase-alpha from regenerating rat liver by the use of a highly sensitive test. This endonuclease activity co-fractionated with DNA polymerase in a great variety of purification procedures involving ion-exchange chromatographies or molecular weight fractionation, but was further completely separated from DNA polymerase activity by using affinity chromatography on DNA-cellulose. The endonuclease acted on native or denatured DNA by introducing single-strand nicks in the DNA molecules; its enzymatic properties indicate that it could act in polymerisation conditions in vitro.     

DNA fragmentation during apoptosis is caused by frequent single-strand cuts.

One of the hallmarks of apoptosis is the digestion of genomic DNA by an endonuclease, generating a ladder of small fragments of double-stranded DNA. We have examined the nature of the DNA breaks produced in mouse thymocytes triggered to undergo apoptosis by steroids or by stimulation of the T cell receptor. Whereas the typical ladder pattern of oligonucleosomal fragments was observed after agarose gel electrophoresis, numerous single-strand cuts were detected after electrophoresis under denaturing conditions. Single-strand nicks were found to be very frequent in the internucleosomal regions, but also to occur in the core particle-associated DNA. An identical pattern of single-strand nicks was obtained when chromatin DNA was exposed to the single-strand cleaving deoxyribonuclease I. The nicked DNA fragments, extracted from apoptotic thymocytes, were sensitive to the action of S1-nuclease. We propose that DNA fragmentation induced during apoptosis is not due to a double-strand cutting enzyme as previously postulated, but rather is the result of single-strand breaks. This ensures the dissociation of the DNA molecule at sites where cuts are found within close proximity.     

Induction and repair of double- and single-strand DNA breaks in bacteriophage lambda superinfecting Escherichia coli

Induction and repair of double- and single-strand DNA breaks have been measured after decays of 125I and 3H incorporated into the DNA and after external irradiation with 4 MeV electrons. For the decay experiments, cells of wild type Escherichia coli K-12 were superinfected with bacteriophage lambda DNA labelled with 5'-(125I)iodo-2'-deoxyuridine or with (methyl-3H)thymidine and frozen in liquid nitrogen. Aliquots were thawed at intervals and lysed at neutral pH, and the phage DNA was assayed for double- and single-strand breakage by neutral sucrose gradient centrifugation. The gradients used allowed measurements of both kinds of breaks in the same gradient. Decays of 125I induced 0.39 single-strand breaks per double-strand break. No repair of either break type could be detected. Each 3H disintegration caused 0.20 single-strand breaks and very few double-strand breaks. The single-strand breaks were rapidly rejoined after the cells were thawed. For irradiation with 4 MeV electrons, cells of wild type E. coli K-12 were superinfected with phage lambda and suspended in growth medium. Irradiation induced 42 single-strand breaks per double-strand break. The rates of break induction were 6.75 x 10(-14) (double-strand breaks) and 2.82 x 10(-12) (single-strand breaks) per rad and per dalton. The single-strand breaks were rapidly repaired upon incubation whereas the double-strand breaks seemed to remain unrepaired. It is concluded that double-strand breaks in superinfecting bacteriophage lambda DNA are repaired to a very small extent, if at all.     

New preparation of PM2 phage DNA and an endonuclease assay for a single-strand break

PM2 is a bacteriophage which has closed circular double-stranded DNA as a genome, which is the sole source for endonuclease assay for a single strand break in the fmol range. Therefore, it is important to isolate PM2 DNA with low control nicks for the endonuclease assay. Usually, the isolation method of phage DNA is to use ultracentrifugation which takes at least 4 days. In this report, a fast and effective method which takes only 2 days was developed to purify DNA using polyethylene glycol (PEG) 8000 and the yields of phage DNA isolated by these two methods were compared. The method using PEG 8000 increased the yield of PM2 DNA from 31.2% to 45.2%, and decreased the nick from 17.1% to 13.1%. Recently, the complete PM2 DNA genome sequence of 10,079 bp was published. The exact number of nucleotides of PM2 DNA is important for the correct enzyme assay which measures nicks generated by an endonuclease. The correct calculation of endonuclease activity of rpS3 for nick-circle assay was performed to measure single-strand breaks in this report. This revised version was published online in June 2006 with corrections to the Cover Date.     

A role for single-strand breaks in bacteriophage phi-X174 genetic recombination

Formation of genetic recombinants in bacteriophage φX174 is stimulated up to 50-fold in host cells carrying the recA⁺ allele by subjecting the virus particles to ultraviolet irradiation before infection, or by starving the host cell for thymine during infection; in recA host strains no such increases are observed.φX174 replicative form DNA molecules formed in vivo from ultraviolet-irradiated bacteriophage consist of an intact, circular full-length viral (+) strand and a partially complete complementary (?) strand extending from the point of origin of complementary strand DNA synthesis to an ultraviolet lesion. φX174 replicative form DNA molecules formed in thymine-deficient host strains during thymine starvation have nearly complete circular viral (+) and complementary (?) strands, which contain random single-strand nicks or gaps.Correlation of these structures with the observed increases in recombination suggests that single-strand “breaks” are aggressive intermediate structures in the formation of φX174 genetic recombinants mediated by the host recA⁺ gene product.     

Adriamycin-mediated introduction of a limited number of single-strand breaks into supercoiled DNA

It was reported previously that Adriamycin converts form I covalently closed circular, supercoiled bacteriophage PM2 DNA to the relaxed circular form II DNA; no form III linear DNA was produced as a result of the extracellular action of Adriamycin in the presence of NADH-dehydrogenase. When form II DNA, produced by the action of Adriamycin, was treated with the BAL 31 nuclease, a single sharp DNA band after agarose gel electrophoresis indicated the presence of only full-length linear form III DNA. As one of its activities, the BAL 31 nuclease introduces a single-strand break in the complementary strand opposite a preexisting single-strand break. When form II DNA, produced by the action of gamma irradiation, was reacted with the BAL enzyme, the resulting linear DNA molecules exhibited a broad range of molecular weights, indicating the presence of many single-strand breaks in the substrate form II DNA. When the Adriamycin-produced form II DNA was treated with restriction endonucleases that cleave PM2 DNA at a single site, either with or without pretreatment with the BAL enzyme, the formation of only full-length linear DNA was observed. Thus, the drug is capable of introducing one or only a very limited number of single-strand breaks into supercoiled DNA; furthermore, these breaks are introduced at random sites along the DNA molecules.     

A mammalian nicking endonuclease.

Purification and properties are described for an endonuclease isolated from calf thymus which attacks double-stranded, unmodified DNA, primarily by making single-strand breaks. No detectable acid-soluble products arise from the reaction. Double-strand breaks may occasionally be produced by the introduction of single-strand breaks on opposite strands in close proximity. The enzyme does not attack denatured DNA and is not inhibited by tRNA. Although added divalent cations are not required for activity, the enzyme is inhibited by EDTA, which suggests an essential role for bound cations; reaction is inhibited by Ca2+. The endonuclease has a broad pH optimum and is inactivated by preincubation at temperatures of 45 degrees C and higher. The molecular weight as determined by gel chromatography is about 30 000. Analysis of the products of reaction on a defined substrate, bacteriophage T3 DNA, by sedimentation in alkaline sucrose density gradients indicates limit products with chain lengths of about 0.8 X 10(6) daltons. On electrophoresis in agarose gels these products were shown to be heterogeneous in size. The endonuclease appears to generate 3'-hydroxyl and 5'-phosphate ends. The ability of the endonuclease to utilize bovine DNA as substrate argues against a restriction role for this enzyme.     

Contribution of topoisomerase I to conversion of single-strand into double-strand DNA breaks

An in vitro system composed of nicked pBR322 DNA and purified topoisomerase I was employed to study the efficiency of the topoisomerase I-driven single-strand to double-strand DNA breaks conversion. At 1.4 × 10⁵ topoisomerase I activity units per mg DNA about 20% single-strand nicks were converted into double-strand breaks during 30 min due to topoisomerase I action. Camptothecin inhibited the conversion. The conversion was also inhibited when the relaxing activity of the used topoisomerase I was increased by phosphorylation of the enzyme with casein kinase 2. The presented data suggest that topoisomerase I may be involved in production of double-stranded breaks in irradiated cells and that this process positively depends on the amount of topoisomerase I but not on its phosphorylation state.     

Bacteriophages of Pseudomonas putida containing single-stranded canonical DNA breaks

It was shown that bacteriophage tf as well as bacteriophages phi p4/40, phi p25/42, phi p23/40 and phi p6/40, which are specific to different P. putida strains, contain the single strand breaks in their DNA. The breaks are localized in one strand of DNA molecules and are repairable with T4 DNA ligase. Bacteriophage tf has no detectable DNA homology with phi p4/40, phi p25/42, phi p23/40 and phi p6/40 bacteriophages. All the phages studied have no relation with other known Pseudomonas phages. Bacteriophages phi p4/40 and phi p25/42 share the extensive DNA homology.     

Chromatin Collapse during Caspase-dependent Apoptotic Cell Death Requires DNA Fragmentation Factor, 40-kDa Subunit-/Caspase-activated Deoxyribonuclease-mediated 3′-OH Single-strand DNA Breaks

Apoptotic nuclear morphology and oligonucleosomal double-strand DNA fragments (also known as DNA ladder) are considered the hallmarks of apoptotic cell death. From a classic point of view, these two processes occur concomitantly. Once activated, DNA fragmentation factor, 40-kDa subunit (DFF40)/caspase-activated DNase (CAD) endonuclease hydrolyzes the DNA into oligonucleosomal-size pieces, facilitating the chromatin package. However, the dogma that the apoptotic nuclear morphology depends on DNA fragmentation has been questioned. Here, we use different cellular models, including MEF CAD^−/− cells, to unravel the mechanism by which DFF40/CAD influences chromatin condensation and nuclear collapse during apoptosis. Upon apoptotic insult, SK-N-AS cells display caspase-dependent apoptotic nuclear alterations in the absence of internucleosomal DNA degradation. The overexpression of a wild-type form of DFF40/CAD endonuclease, but not of different catalytic-null mutants, restores the cellular ability to degrade the chromatin into oligonucleosomal-length fragments. We show that apoptotic nuclear collapse requires a 3′-OH endonucleolytic activity even though the internucleosomal DNA degradation is impaired. Moreover, alkaline unwinding electrophoresis and In Situ End-Labeling (ISEL)/In Situ Nick Translation (ISNT) assays reveal that the apoptotic DNA damage observed in the DNA ladder-deficient SK-N-AS cells is characterized by the presence of single-strand nicks/breaks. Apoptotic single-strand breaks can be impaired by DFF40/CAD knockdown, abrogating nuclear collapse and disassembly. In conclusion, the highest order of chromatin compaction observed in the later steps of caspase-dependent apoptosis relies on DFF40/CAD-mediated DNA damage by generating 3′-OH ends in single-strand rather than double-strand DNA nicks/breaks.     

Ddc1 checkpoint protein and DNA polymerase ɛ interact with nick-containing DNA repair intermediate in cell free extracts of Saccharomyces cerevisiae

To characterize proteins that interact with base excision/single-strand interruption repair DNA intermediates in cell free extracts of Saccharomyces cerevisiae, we used a combination of photoaffinity labeling with the protein identification by MALDI-TOF-MS peptide mapping. Photoreactive analogue of dCTP, namely exo-N-[4-(4-azido-2,3,5,6,-tetrafluorobenzylidenehydrazinocarbonyl)-butylcarbamoyl]-2'-deoxycytidine-5'-triphosphate, and [(32)P]-labeled DNA duplex containing one nucleotide gap were used to generate nick-containing DNA with a photoreactive dCMP residue at the 3'-margin of the nick. This photoreactive DNA derivative was incubated with the yeast cell extract and after UV irradiation a number of proteins were labeled. Two of the crosslinked proteins were identified as the catalytic subunit of DNA polymerase ? and Ddc1 checkpoint protein. Labeling of DNA polymerase ? catalytic subunit with the nick-containing DNA repair intermediate indicates that the DNA polymerase is involved in the DNA repair synthesis in yeast, at least at DNA single-strand interruptions. Crosslinking of Ddc1 to DNA nicks took place independently of the other components of checkpoint clamp, Mec3 and Rad17, suggesting that the protein alone is able to recognize DNA single-strand breaks. Indeed, purified GST-tagged Ddc1 protein was efficiently crosslinked to nick-containing DNA. The interaction of Ddc1 with DNA nicks may provide a link between the DNA damage checkpoint and DNA base excision/single-strand breaks repair pathways in yeast. In addition, we found that absence of Ddc1 protein greatly influences the overall pattern of other proteins crosslinked to DNA nick. We suggested that this last effect of Ddc1 is at least partially due to its capacity to prevent proteolytic degradation of the DNA-protein adducts.     

Processing of model single-strand breaks in phi X-174 RF transfecting DNA by Escherichia coli

The inactivation efficiency and repair of single-strand breaks was investigated using model strand breaks created by endonucleolytic incision of damaged DNA. Phi X-174 duplex transfecting DNA containing either thymine glycols, urea residues, or abasic (AP) sites was incubated with AP endonucleases that produce breaks on the 3' side, the 5' side, or both sides of the lesion. For each lesion, incubation with Escherichia coli endonuclease III results in a single-strand break containing a 3' alpha, beta-unsaturated aldehyde (4-hydroxy-2-pentenal), while treatment of AP- or urea-containing DNA with E. coli endonuclease IV results in a single-strand break containing a 5' deoxyribose or a 5' deoxyribosylurea moiety, respectively. Incubation of lesion-containing DNA with both enzymes results in a base gap. Ligatable nicks containing 3' hydroxyl and 5' phosphate moieties were produced by subjecting undamaged DNA to DNase I. When the biological activity of these DNAs was assessed in wild-type cells, ligatable nicks were not lethal, but each of the other strand breaks tested was lethal, having inactivation efficiencies between 0.12 and 0.14. These inactivation efficiencies are similar to those of the base lesions from which the strand breaks were derived. In keeping with the current model of base excision repair, when phi X duplex DNA containing strand breaks with a blocked 3' terminus was transfected into an E. coli double mutant lacking the major 5' cellular AP endonucleases, a greater than twofold decrease in survival was observed. Moreover, when this DNA was treated with a 5' AP endonuclease prior to transfection, the survival returned to that of wild type. As expected, when DNA containing strand breaks with a 5' blocked terminus or DNA containing base gaps was transfected into the double mutant lacking 5' AP endonucleases, the survival was the same as in wild-type cells. The decreased survival of transfecting DNA containing thymine glycols, urea, or AP sites observed in appropriate base excision repair-defective mutants was also obviated if the DNA was incubated with the homologous enzyme prior to transfection. Thus, in every case, with both base lesions and single-strand breaks, the lesion was repaired in the cell by the enzyme that recognizes it in vitro. Furthermore, the repair step in the cell could be eliminated if the appropriate enzyme was added in vitro prior to transfection.(ABSTRACT TRUNCATED AT 400 WORDS)