Two classes of single-stranded regions evident in deproteinized preparations of replicating DNA isolated from mammalian cells

In DNA isolated from proliferating human lymphoblastoid CCRF-CEM cells which had been pulse-labeled by exposure to [3H]thymidine for periods from 30 s to 10 min, single-stranded regions were analyzed by caffeine-gradient elution from benzoylated DEAE-cellulose. Two classes of structural defect were evident. Some replicating DNA exhibited single-stranded regions of approximately 200 nucleotides, while most newly incorporated radioactivity was associated with DNA containing single-stranded regions from 900 to approximately 4000 nucleotides. The distribution of thymidine-derived radioactivity did not suggest sequential or preferential labeling of these DNA fractions as the incorporation time was varied. The findings may be correlated with recent proposals regarding the structural basis of eukaryotic DNA replication.     

Extensive regions of single-stranded DNA in aphidicolin-treated melanoma cells

We have looked for the presence of single-stranded DNA in human melanoma cells. Single-stranded DNA was observed by lysis of cells in dilute alkali (to partly denature the DNA) followed by CsCl gradient centrifugations. In normally growing cells we did not observe single-stranded DNA whereas large amounts were present in cells treated with aphidicolin (an inhibitor of DNA polymerase alpha). The single-stranded DNA is much larger (greater than 20 kb) than Okazaki fragments. When the cells were washed free of aphidicolin, the single-stranded DNA was converted to high molecular weight DNA. Furthermore, when DNA synthesis is recovering after drug treatment, the single-stranded DNA disappears. The single-stranded DNA represents a transient step during the maturation of newly synthesized DNA.     

The origin of nascent single-stranded DNA extracted from mammalian cells.

In vitro cultured bovine liver cells were labelled with radioactive thymidine and dissolved in 0.5% sodium dodecyl sulphate. Centrifugation of the lysate through sucrose gradients in a zonal rotor revealed a slowly sedimenting fraction of preferentially pulse labelled DNA. The DNA of this zone was further analysed by chromatography on hydroxy-apatite, banding in CsCl density gradients, and sedimentation in neutral and alkaline sucrose gradients. It contained besides small amounts of fragmented bulk DNA, single-stranded nascent DNA and single-stranded pre-labelled DNA which could be separated from each other by using BrdU as a density label. The density labelling also revealed small amounts of nascent-nascent DNA duplexes. The slowly sedimenting fraction was practically absent from cell lysates which were prepared in 2 M NaCl - 50 microgram/ml pronase. The results suggest that nascent single-strands and nascent-nascent duplexes are released from the forks of replicating DNA by branch migration. Pre-labelled single strands may be released by the same branch migration. Pre-labelled single strands may be released by the same mechanism, but the in vivo structure from which they originate has yet to be elucidated.     

Targeted recombination with single-stranded DNA vectors in mammalian cells.

We studied the ability of single-stranded DNA (ssDNA) to participate in targeted recombination in mammalian cells. A 5' end-deleted adenine phosphoribosyltransferase (aprt) gene was subcloned into M13 vector, and the resulting ssDNA and its double-stranded DNA (dsDNA) were transfected to APRT-Chinese hamster ovary cells with a deleted aprt gene. APRT+ recombinants with the ssDNA was obtained at a frequency of 3 x 10(-7) per survivor, which was almost equal to that with the double-stranded equivalent. Analysis of the genome in recombinant clones produced by ssDNA revealed that 12 of 14 clones resulted from correction of the deletion in the aprt locus. On the other hand, the locus of the remaining 2 was not corrected; instead, the 5' deletion of the vector was corrected by end extension, followed by integration into random sites of the genome. To exclude the possibility that input ssDNA was converted into its duplex form before participating in a recombination reaction, we compared the frequency of extrachromosomal recombination between noncomplementary ssDNAs, and between one ssDNA and one dsDNA, of two phage vectors. The frequency with the ssDNAs was 0.4 x 10(-5), being 10-fold lower than that observed with the ssDNA and the dsDNA, suggesting that as little as 10% of the transfected ssDNA was converted into duplex forms before the recombination event, hence 90% remained unchanged as single-stranded molecules. Nevertheless, the above finding that ssDNA was as efficient as dsDNA in targeted recombination suggests that ssDNA itself is able to participate directly in targeted recombination reactions in mammalian cells.     

Deoxyadenylate-rich and deoxyguanylate-rich regions in mammalian DNA

The presence of deoxyadenylate-rich and deoxyguanylate-rich regions in mammalian DNA has been demonstrated by hybridization with ³H-labelled poly(U) and ³H-labelled poly(C). For hamster BHK-21/C13 cells, the dA-rich regions are up to 130 nucleotides long and comprise up to 0.4% of the DNA. Those dA-rich regions which comprise 0.13% of the DNA contain 2 to 6% of bases other than adenine. The dG-rich regions, in which 10 to 30% of the bases are other than guanine, are less than 40 nucleotides long and are present at a level of about 0.1% of the DNA. Exhaustive digestion of the hybrids with RNAase enables detection of deoxyhomopolymeric regions in the DNA, poly (dA) sequences of an average size of about 30 nucleotides long accounting for 0.008% of the DNA, and poly(dG) sequences, 17 nucleotides long, comprising 0.0016% of the DNA.Both dA-rich and dG-rich regions are found in DNA sequences with a wide variety of base composition. Extensive shearing of the DNA is required to produce some enrichment for dA-rich sequences in the (A + T)-rich fraction, although dG-rich sequences are slightly enriched in the (G + C)-rich fraction of even unsheared DNA. The buoyant density of hybrid molecules was found to be significantly greater than that of unhybridized DNA only when highly sheared DNA was used. These findings suggest that the dA-rich and dG-rich regions have a widespread distribution throughout DNA molecules. In situ hybridization studies with ³H-labelled poly(U) further suggest that the dA-rich regions are not localized to any particular chromosome or to any specific region of the chromosomes. Analysis of DNA from a number of different species has shown that, in general, the dA-rich and dG-rich regions are present at a much higher level in mammalian DNA than in bacterial, bacteriophage or mammalian virus DNA.Possible functions of these unusual deoxynucleotide sequences are discussed.     

Sizing of single-stranded regions in double-stranded DNA by preparative benzoylated DEAE-cellulose chromatography

The concentration of caffeine required to elute wholly single-stranded DNA from benzoylated DEAE-cellulose is proportional to the polynucleotide length. The use of benzoylated DEAE-cellulose chromatography for isolating and sizing single-stranded regions in double-stranded DNA has been examined using a series of hybrid molecules. Restriction fragments of the replicating form of bacteriophage luminal diameter X174 were hybridized to the intact 'plus' strand, thereby forming hybrids having single- and/or double-stranded regions in the kilobase range. A series of such hybrid preparations were subject to caffeine concentration gradient elution from benzoylated DEAE-cellulose. After logarithmic transformation, a linear relationship (R = 0.94) could be demonstrated between eluting caffeine concentration and single-stranded length, irrespective of the length of associated double-stranded regions or the location, within a given fragment, of unpaired nucleotides. Benzoylated DEAE-cellulose chromatography may therefore be used to separate and characterize, on a preparative scale, double-stranded DNA containing single-stranded regions.     

Direct measurement of single-stranded DNA intermediates in mammalian cells by quantitative polymerase chain reaction

Single-stranded DNA (ssDNA) intermediates are formed in multiple cellular processes, including DNA replication and recombination. Here, we describe a quantitative polymerase chain reaction (qPCR)-based assay to quantitate ssDNA intermediates, specifically the 3′ ssDNA product of resection at specific DNA double-strand breaks induced by the AsiSI restriction enzyme in human cells. We protect the large mammalian genome from shearing by embedding the cells in low-gelling-point agar during genomic DNA extraction and measure the levels of ssDNA intermediates by qPCR following restriction enzyme digestion. This assay is more quantitative and precise compared with existing immunofluorescence-based methods.     

Repair of single-stranded DNA nicks, gaps, and loops in mammalian cells.

We studied the ability of mammalian cells to repair single-stranded nicks, gaps, and loops in DNA duplexes. Heteroduplexes prepared from derivatives of the shuttle vector pSV2neo were introduced into monkey COS cells. After replication, the plasmids were recovered and used to transform Escherichia coli. Plasmid DNA from the recovered colonies was tested for repair at each of six different sites. We observed that mammalian cells are capable of repairing single-stranded gaps and free single-stranded ends most efficiently. Regions containing twin loops were recognized, and one of the loops was excised. Portions of the molecules containing small single loops were also repaired. Markers which were 58 nucleotides apart were corepaired with nearly 100% efficiency, while markers which were 1,000 nucleotides or more apart were never corepaired. The mechanisms involved in heteroduplex repair in mammalian cells seem to be similar to those involved in repairing DNA lesions caused by physical and chemical agents.     

The relation of single-stranded regions in bacteriophage PM2 supercoiled DNA to the early melting sequences.

Bacteriophage PM2 supercoiled DNA contains one to three small single-stranded regions that can be detected in the electron microscope after various treatments. The relative positions of these regions were mapped against the unique cleavage site for the restriction endonuclease R · HapII on PM2 DNA. Any of eight sharply defined regions of the genome may be single-stranded in supercoiled molecules. They are found in all possible combinations of three or less and at approximately the same frequency. A comparison of this map of supercoiled DNA with the alkaline denaturation pattern of nicked circular or linear PM2 DNA showed that these same regions were also the earliest melting regions in non-supercoiled DNA.     

An ATPase depending on the presence of single-stranded DNA from mouse myeloma.

An ATPase was purified from mouse myeloma MOPC 70E the activity of which depends on the presence of single-stranded DNA and divalent cations such as Mg2+, Mn2+, Ca2+, Ni2+ or Fe2+. The enzyme splits both ribonucleoside and deoxyribonucleoside triphosphates but preferentially ATP and dATP yielding nucleoside diphosphates and inorganic phosphate. The enzyme has an absolute requirement for single-stranded DNA. Alternating double-stranded polydeoxynucleotides are only slight effective, and native double-stranded DNA, single-stranded and double-stranded RNAs as well as DNA - RNA hybrids are ineffective in stimulating the ATPase. The enzyme has further characterized by sedimentation in a sucrose density gradient (s20, w = 5.5 S) and by isoelectric focussing in an ampholine pH gradient (pI = 6.5).     

Cloning single-stranded DNA

DNA sequence and expression analyses have greatly benefited from using M13 and pUC derived cloning vectors and their polycloning sites. A chronology of the original concepts and experiments is reviewed.     

The metaphase chromosome: functional inertia and ability related to the presence of sequences of single-stranded DNA

Isolated metaphase chromosomes from KB cells were used as template in an in vitro DNA synthesis assay. In these conditions, no synthesis was noticed, confirming template inactivity of isolated metaphase chromosomes. DNA synthesis was noticed after a pretreatment with either methanol-acetic acid or RNase A. Analysis of in vitro synthesized polydeoxyribonucleotides showed two fractions of 4 S and 7-8 S. These results suggest the presence in metaphase chromosome of single stranded DNA sequences. Such sequences are shown in DNA extracted from chromosomes. They would preexist in this organelle and would be unmasked by the treatments that restore template activity of metaphase chromosome.     

Subtelomeric regions in mammalian cells are deficient in DNA double-strand break repair

We have previously demonstrated that double-strand breaks (DSBs) in regions near telomeres are much more likely to result in large deletions, gross chromosome rearrangements, and chromosome instability than DSBs at interstitial sites within chromosomes. In the present study, we investigated whether this response of subtelomeric regions to DSBs is a result of a deficiency in DSB repair by comparing the frequency of homologous recombination repair (HRR) and nonhomologous end joining (NHEJ) at interstitial and telomeric sites following the introduction of DSBs by I-SceI endonuclease. We also monitored the frequency of small deletions, which have been shown to be the most common mutation at I-SceI-induced DSBs at interstitial sites. We observed no difference in the frequency of small deletions or HRR at interstitial and subtelomeric DSBs. However, the frequency of NHEJ was significantly lower at DSBs near telomeres compared to interstitial sites. The frequency of NHEJ was also lower at DSBs occurring at interstitial sites containing telomeric repeat sequences. We propose that regions near telomeres are deficient in classical NHEJ as a result of the presence of cis-acting telomere-binding proteins that cause DSBs to be processed as though they were telomeres, resulting in excessive resection, telomere loss, and eventual chromosome rearrangements by alternative NHEJ.     

The frequency of oligonucleotides in mammalian genic regions

The large body of nucleic acid sequence data now available offersa unique opportunity for the characterization of individualoligonucleotides which may be specific to sequence functionaldomains. We have prepared algorithms for the study of the frequencydistribution of all oligonucleotides of length 2–6 inDNA sequences. We have implemented them in the study of 634mammalian DNA sequences spanning 1.782 Mb, and have obtainedthe distribution of the ratio between the observed frequencyof oligonucleotides and their expected frequency based on independentnucleotide probabilities. We then studied the distribution ofoligonucleotides (or k-tuples) of each length in a subset of129 complete mammalian genes spanning 0.607 Mb. Eight distinctgenomic regions, namely 5'-non-transcribed, first exon, firstintron, intermediate exons, intermediate introns, last intron,last exon and 3non-transcribed, were considered. We observedthat some oligonucleotides show a statistical behaviour anda regional distribution similar to that of known signal sequences.Moreover the frequency distribution of oligonucleotides of length5 and 6 tends to become bimodal, indicating the existence ofa population of very frequent oligonucleotides. Received on June 21, 1988; accepted on October 14, 1988     

Formation of single-stranded regions in the course of digestion of DNA with DNAase II and micrococcal nuclease

In the course of digestion of DNA with DNAase II or micrococcal nuclease, considerable amounts of single-stranded (ss) regions are formed, as determined by a second digestion with ss-specific nucleases, hyperchromicity measurements, and electron microscopy. Most of the ss stretches are located internally in the DNA molecules. The effect appears to be related to regions of decreased stability arising around single-stranded cuts in the double helix.