Cloning full-length, cap-trapper-selected cDNAs by using the single-strand linker ligation method.

We have developed the single-strand linker ligation method (SSLLM), which uses DNA ligase to add a dsDNA linker to single-stranded (ss) full-length cDNA. The linkers have random 6-bp (dN6 or dGN5) 3' overhangs that can ligate to any cDNA sequence, thereby facilitating the production of cDNA libraries with titers exceeding 1 x 10(6) independent clones. We confirmed that the 5' ends of cDNA inserts cloned by using SSLLM are full-length and include the 5' untranslated regions. The great advantage of our method is that the elimination of the GC tail simplifies the sequencing and protein translation of the full-length clones. Further, our method tags ss cDNAs more efficiently than does the traditional RNA ligase reaction.     

Conversion of phosphoglycolate to phosphate termini on 3' overhangs of DNA double strand breaks by the human tyrosyl-DNA phosphodiesterase hTdp1

Mammalian cells contain potent activity for removal of 3'-phosphoglycolates from single-stranded oligomers and from 3' overhangs of DNA double strand breaks, but no specific enzyme has been implicated in such removal. Fractionated human whole-cell extracts contained an activity, which in the presence of EDTA, catalyzed removal of glycolate from phosphoglycolate at a single-stranded 3' terminus to leave a 3'-phosphate, reminiscent of the human tyrosyl-DNA phosphodiesterase hTdp1. Recombinant hTdp1, as well as Saccharomyces cerevisiae Tdp1, catalyzed similar removal of glycolate, although less efficiently than removal of tyrosine. Moreover, glycolate-removing activity could be immunodepleted from the fractionated extracts by antiserum to hTdp1. When a plasmid containing a double strand break with a 3'-phosphoglycolate on a 3-base 3' overhang was incubated in human cell extracts, phosphoglycolate processing proceeded rapidly for the first few minutes but then slowed dramatically, suggesting that the single-stranded overhangs gradually became sequestered and inaccessible to hTdp1. The results suggest a role for hTdp1 in repair of free radical-mediated DNA double strand breaks bearing terminally blocked 3' overhangs.     

Transient generation of displaced single-stranded DNA during nick translation

We show that displaced single-stranded overhangs are transiently generated and destroyed during nick translation by E. coli DNA polymerase I. Evidence that hyper-rec mutants have an increased frequency of such overhang structures is discussed. The transient generation of overhangs may be significant for general recombination. The 5' leads to 3' exonuclease activity of polymerase I specifically hydrolyzes such overhangs to yield a nick. Overhangs are generated by polymerization, but after every polymerization step, either polymerase or exonuclease can act--55% of the time, polymerization occurred first. At this frequency overhangs of greater than or equal to 12 nucleotides are generated every 1300 nucleotides polymerized. We suggest that many DNA strand discontinuities are displaced single-stranded overhangs, rather than gaps or simple nicks.     

Presence of double-strand breaks with single-base 3' overhangs in cells undergoing apoptosis but not necrosis

Apoptotic cells in rat thymus were labeled in situ in paraffin-embedded and frozen tissue sections by ligation of double-stranded DNA fragments containing digoxigenin or Texas red. Two forms of double-stranded DNA fragments were prepared using the polymerase chain reaction: one was synthesized using Taq polymerase, which yields products with single- base 3' overhangs, and one using Pfu polymerase, which produces blunt- ended products. Both types of fragment could be ligated to apoptotic nuclei in thymus, indicating the presence in such nuclei of DNA double- strand breaks with single-base 3' overhangs as well as blunt ends. However, in nuclei with DNA damage resulting from a variety of nonapoptotic processes (necrosis, in vitro autolysis, peroxide damage, and heating) single-base 3' overhangs were either nondetectable or present at much lower concentrations than in apoptotic cells. Blunt DNA ends were present in such tissues, but at lower concentrations than in apoptotic cells. In contrast, in all of these forms of DNA damage, nuclei contained abundant 3'-hydroxyls accessible to labeling with terminal deoxynucleotidyl transferase. Thus, although single-base 3' overhangs and blunt ends are present in apoptotic nuclei, the specificity of the in situ ligation of 3'-overhang fragments to apoptotic nuclei indicates that apoptotic cells labeled in this way can readily be distinguished from cells with nonapoptotic DNA damage. These data are consistent with the involvement of an endonuclease similar to DNase I in apoptosis, which is predicted to leave short 3' overhangs as well as blunt ends in digestion of chromatin.     

Relative affinities of poly(ADP-ribose) polymerase and DNA-dependent protein kinase for DNA strand interruptions

Poly(ADP-ribose) polymerase (PARP) and DNA-dependent protein kinase (DNA-PK) are important nuclear enzymes that cooperate to minimize genomic damage caused by DNA strand interruptions. DNA strand interruptions trigger the ADP-ribosylation activity and phosphorylation activity of PARP and DNA-PK respectively. In order to understand the relationship of PARP and DNA-PK with respect to DNA binding required for their activation, we analyzed the kinetics of the reactions and determined the apparent dissociation constants (Kd app) of the enzymes for DNA strand interruptions. PARP has a high binding affinity for blunt ends of DNA (Kd app=116 pM) and 3' single-base overhangs (Kd app=332 pM) in comparison to long overhangs (Kd app=2.6-5.0 nM). Nicks are good activators of PARP although the affinity of PARP for nicks (Kd app=467 pM) is 4-fold less than that for blunt ends. The Kd app of DNA-PK for 3' single-base overhangs, blunt ends and long overhangs is 704 pM, 1.3 nM and 1.4-2.2 nM respectively. These results demonstrate that (1) PARP, when compared to DNA-PK, has a greater preference for blunt ends and 3' single-base overhangs but a weaker preference for long overhangs, and (2) nicks are effective in attracting and activating PARP. The possible implications of the preferences of PARP and DNA-PK for DNA strand interruptions in vivo are discussed.     

The generation of long telomere overhangs in human cells: a model and its implication

MOTIVATION: Linear chromosomes carry on both ends repetitive DNA sequences called telomere. In the conventional model of semi-conservative DNA replication, the 3'-end of a linear DNA strand cannot be fully replicated, resulting in a single-stranded 3' overhang at one end of the double-stranded DNA product. In this model, the length of the overhang is expected to be about the size of an RNA primer (about nine nucleotides for human cells). However, it has been found that the telomere overhangs in human cells can be as long as several hundred nucleotides. At present, the opinion regarding how such long overhangs are produced is controversial. RESULTS: In order to gain insight into the mechanism by which long telomere overhangs are produced, we derived a mathematical model that can perfectly describe the length distribution of telomere overhangs in several human cell strains. The model suggests that the production of telomere overhangs can be explained by three contributions corresponding to three regions on the G-rich telomere template strand, namely, the region occupied by the last primer, that missed out by this primer at its 5'-side and the 3'-terminus of the template strand that is inaccessible to primase. The model can also be used to simulate incomplete telomere replication.     

Seamless gene engineering using RNA- and DNA-overhang cloning

Here we describe two methods for generating DNA fragments with single-stranded overhangs, like those generated by the activity of many restriction enzymes, by simple methods that do not involve DNA digestion. The methods, RNA-overhang cloning (ROC) and DNA-overhang cloning (DOC), generate polymerase chain reaction (PCR) products composed of double-stranded (ds) DNA flanked by single-stranded (ss) RNA or DNA overhangs. The overhangs can be used to recombine DNA fragments at any sequence location, creating "perfect" chimeric genes composed of DNA fragments that have been joined without the insertion, deletion, or alteration of even a single base pair. The ROC method entails using PCR primers that contain regions of RNA sequence that cannot be copied by certain thermostable DNA polymerases. Using such a chimeric primer in PCR would yield a product with a 5' overhang identical to the sequence of the RNA component of the primer, which can be used for directional ligation of the amplified product to other preselected DNA molecules. This method provides complete control over both the length and sequence of the overhangs, and eliminates the need for restriction enzymes as tools for gene engineering.     

Processing of 3'-phosphoglycolate-terminated DNA double strand breaks by Artemis nuclease

The Artemis nuclease is required for V(D)J recombination and for repair of an as yet undefined subset of radiation-induced DNA double strand breaks. To assess the possibility that Artemis acts on oxidatively modified double strand break termini, its activity toward model DNA substrates, bearing either 3'-hydroxyl or 3'-phosphoglycolate moieties, was examined. A 3'-phosphoglycolate had little effect on Artemis-mediated trimming of long 3' overhangs (> or =9 nucleotides), which were efficiently trimmed to 4-5 nucleotides. However, 3'-phosphoglycolates on overhangs of 4-5 bases promoted Artemis-mediated removal of a single 3'-terminal nucleotide, while at least 2 nucleotides were trimmed from identical hydroxyl-terminated substrates. Artemis also efficiently removed a single nucleotide from a phosphoglycolate-terminated 3-base 3' overhang, while leaving an analogous hydroxyl-terminated overhang largely intact. Such removal was completely dependent on DNA-dependent protein kinase and ATP and was largely dependent on Ku, which markedly stimulated Artemis activity toward all 3' overhangs. Together, these data suggest that efficient Artemis-mediated cleavage of 3' overhangs requires a minimum of 2 nucleotides, or a nucleotide plus a phosphoglycolate, 3' to the cleavage site, as well as 2 unpaired nucleotides 5' to the cleavage site. Shorter 3'-phosphoglycolate-terminated overhangs and blunt ends were also processed by Artemis but much more slowly. Consistent with a role for Artemis in repair of terminally blocked double strand breaks in vivo, human cells lacking Artemis exhibited hypersensitivity to x-rays, bleomycin, and neocarzinostatin, which all induce 3'-phosphoglycolate-terminated double strand breaks.     

Analysis of non-template-directed nucleotide addition and template switching by DNA polymerase

DNA polymerases use an uninterrupted template strand to direct synthesis of DNA. However, some DNA polymerases can synthesize DNA across two discontinuous templates by binding and juxtaposing them, resulting in synthesis across the junction. Primer/template duplexes with 3' overhangs are especially efficient substrates, suggesting that DNA polymerases use the overhangs as regions of microhomology for template synapsis. The formation of these overhangs may be the result of non-template-directed nucleotide addition by DNA polymerases. To examine the relative magnitude and mechanism of template switching, we studied the in vitro enzyme kinetics of template switching and non-template-directed nucleotide addition by the 3'-5' exonuclease-deficient large fragment of Escherichia coli DNA polymerase I. Non-template-directed nucleotide addition and template switching were compared to that of standard primer extension. We found that non-template-directed nucleotide addition and template switching showed similar rates and were approximately 100-fold slower than normal template-directed DNA synthesis. Furthermore, non-template-directed nucleotide addition showed a 10-fold preference for adding dAMP to the ends of DNA over that of the other three nucleotides. For template switching, kinetic analysis revealed that the two template substrates acted as a random bireactant system with mixed-type inhibition of substrate binding by one substrate over the other. These data are the first to establish the binding kinetics of two discontinuous DNA substrates to a single DNA polymerase. Our results suggest that although the activities are relatively weak, non-template-directed nucleotide addition and template switching allow DNA polymerases to overcome breaks in the template strand in an error-prone manner.     

Pol3 is involved in nonhomologous end-joining in Saccharomyces cerevisiae

Nonhomologous end joining connects DNA ends in the absence of extended sequence homology and requires removal of mismatched DNA ends and gap-filling synthesis prior to a religation step. Pol4 within the Pol X family is the only polymerase known to be involved in end processing during nonhomologous end joining in yeast. The Saccharomyces cerevisiae POL3/CDC2 gene encodes polymerase delta that is involved in DNA replication and other DNA repair processes. Here, we show that POL3 is involved in nonhomologous end joining using a plasmid-based end-joining assay in yeast, in which the pol3-t mutation caused a 1.9- to 3.2-fold decrease in the end-joining efficiency of partially compatible 5' or 3' ends, or incompatible ends, similar to the pol4 mutant. The pol3-t pol4 double mutation showed a synergistic decrease in the efficiency of NHEJ with partially compatible 5' ends or incompatible ends. Sequence analysis of the rejoined junctions recovered from the wild-type cells and mutants indicated that POL3 is required for gap filling at 3' overhangs, but not 5' overhangs during POL4-independent nonhomologous end joining. We also show that either Pol3 or Pol4 is required for simple religation of compatible or blunt ends. These results suggest that Pol3 has a generalized function in end joining in addition to its role in gap filling at 3' overhangs to enhance the overall efficiency of nonhomologous end joining. Moreover, the decreased end-joining efficiency seen in the pol3-t mutant was not due to S-phase arrest associated with the mutant. Taken together, our genetic evidence supports a novel role of Pol3 in nonhomologous end joining that facilitates gap filling at 3' overhangs in the absence of Pol4 to maintain genomic integrity.     

C. elegans telomeres contain G-strand and C-strand overhangs that are bound by distinct proteins

Single-strand extensions of the G strand of telomeres are known to be critical for chromosome-end protection and length regulation. Here, we report that in C. elegans, chromosome termini possess 3' G-strand overhangs as well as 5' C-strand overhangs. C tails are as abundant as G tails and are generated by a well-regulated process. These two classes of overhangs are bound by two single-stranded DNA binding proteins, CeOB1 and CeOB2, which exhibit specificity for G-rich or C-rich telomeric DNA. Strains of worms deleted for CeOB1 have elongated telomeres as well as extended G tails, whereas CeOB2 deficiency leads to telomere-length heterogeneity. Both CeOB1 and CeOB2 contain OB (oligo-saccharide/oligo-nucleotide binding) folds, which exhibit structural similarity to the second and first OB folds of the mammalian telomere binding protein hPOT1, respectively. Our results suggest that C. elegans telomere homeostasis relies on a novel mechanism that involves 5' and 3' single-stranded termini.     

Telomere shortening in human fibroblasts is not dependent on the size of the telomeric-3'-overhang

Telomeres shorten in human somatic cells with each round of DNA replication, and this shortening is thought to ultimately trigger replicative senescence. Telomere shortening is caused partly by the inability of semiconservative DNA replication to copy a linear strand of DNA to its very end. Post-replicative processing of telomeric ends, producing single-stranded G-rich 3' overhangs, has also been suggested to contribute to telomere shortening. This suggestion implies that a positive correlation should exist between the length of 3' overhangs and the rate of telomere shortening. We confirmed shortening of overhangs as human lung (MRC5) and foreskin (BJ) fibroblasts approach senescence by measuring overhang length using in-gel hybridization. However, a large study of fibroblast strains from 21 donors maintained under conditions which lead to two orders of magnitude of variation in telomere shortening rate failed to show any correlation between telomere overhang length and shortening rate, suggesting that overhang length is neither a cause nor a correlate of telomere shortening.     

A phagemid vector library for cloning DNA with four-nucleotide 5' or 3' overhangs

A phagemid vector library for cloning DNA with four nucleotide 5' or 3' overhangs has been constructed. This library is based on the pT7T3 vector (Pharmacia) which is a modification of the phagemid pTZ18U vector. We have chosen pT7T3 as the parent vector because it can be used for Sanger's dideoxy sequencing and for the generation of RNA probes with either the T7 or T3 promoter. Each member of the cloning vector series pBM has recognition sites for both of the restriction enzymes BspM1 and BstX1 in addition to the basic multiple cloning sites. BspM1 recognizes the sequence 5'...ACCTGC NNNN/NNNN...3' whereas BstX1 recognizes the sequence 5'...CCAN NNNN/NTGG...3'. Thus these two sites can be overlapped, so that only 256 vectors (instead of 512 vectors) need be constructed to cover all the theoretical possible combinations of sites which give complementary cohesive ends for cloning DNA with four nucleotide 5' or 3' overhangs. This vector library can be used for amplification cloning of DNA in a tandem array by choosing appropriate vectors which have nonpalindromic sequences. We have obtained approximately 200 members of the 256 possible clones and have organized the vectors using a MacIntosh HyperCard program for easy retrieval.     

Enzymatic inverse PCR: a restriction site independent, single-fragment method for high-efficiency, site-directed mutagenesis.

A new method is described for rapid site-directed mutagenesis of plasmid DNA. The new method, termed enzymatic inverse polymerase chain reaction (EIPCR), uses inverse PCR to amplify the entire plasmid. The key step to EIPCR is the incorporation of identical class 2s restriction sites in both primers. Class 2s restriction enzymes have a recognition site that is located 5' of the cut site (e.g., BsaI: GGTCTCN'NNNN,). Thus, after completing PCR, the ends of the full-length linearized plasmid are digested with the class 2s enzyme incorporated into the primers. The enzyme cuts off its entire recognition site and leaves the plasmid with compatible overhangs on both ends. Thus, in the ligation the only part that becomes part of the plasmid is the NNNN overhang, which can be made to be the native sequence. We have used the method for many plasmids and several class 2s enzymes. As an example, we report here the use of EIPCR for an insertion into pUC19 containing an inactive lacZ alpha-peptide, causing a frameshift that restores lacZ alpha-activity. Of 300 colonies evaluated, greater than 95% had the expected blue phenotype. The BsaI overhangs were correctly combined in all of the 35 blue colonies analyzed by restriction digestion and in all four clones that were sequenced. EIPCR is compared with four related PCR-based mutagenesis techniques. The major advantage of EIPCR over the other methods is the combination of greater than 95% correctly mutated clones with the need for only two PCR primers.     

Universal restriction site-free cloning method using chimeric primers

A universal restriction site-free cloning method has been developed to precisely insert a DNA fragment into a vector at any desired location without altering any nucleotide(s) in either the DNA fragment or the vector. The technique employs two pairs of chimeric primers, each containing a ribonucleotide. One pair of primers is used to amplify a target DNA fragment and another is used to prepare a linear vector. The ribonucleotide is used as a specific site for cleavage promoted by rare-earth metal ions such as La3+ or Lu3+. Therefore, blunt-ended PCR products can be converted into a dsDNA with single-stranded 3'overhangs for efficient ligation. The primers are designed so that both the target DNA fragment and vector PCR products create defined 3' overhangs to permit the formation of a seamless plasmid during the subsequent ligation. This method has been used successfully to clone the E. coli gene coding for peptidyl-tRNA hydrolase.     

Studies of oligonucleotide interactions by hybridisation to arrays: the influence of dangling ends on duplex yield.

Effects of dangling ends on duplex yield have been assessed by hybridisation of oligonucleotides to an array of oligonucleotides synthesised on the surface of a solid support. The array consists of decanucleotides and shorter sequences. One of the decanucleotides in the array was fully complementary to the decanucleotide used as solution target. Others were complementary over seven to nine bases, with overhangs of one to three bases. Duplexes involving different decanucleotides had different overhangs at the 3' and 5' ends. Some duplexes involving shorter oligonucleotides had the same regions of complementarity as these decanucleotides, but with fewer overhanging bases. This analysis allows simultaneous assessment of the effects of differing bases at both 5' and 3' ends of the oligonucleotide in duplexes formed under identical reaction conditions. The results indicate that a 5' overhang is more stabilising than a 3' overhang, which is consistent with previous results obtained with DNA overhangs. However, it is not clear whether this is due to the orientation of the overhang or to the effect of specific bases.     

Self-assembly cloning: a rapid construction method for recombinant molecules from multiple fragments

Enzyme-free cloning (EFC) can rapidly produce an in-frame fusion gene with multiple fragments. To practically apply EFC, we investigated the extent and sequence of complementary staggered overhangs necessary to direct self-assembly of multiple fragments as well as a size limitation of the constructed DNA molecule. Six-base pair overhangs with 50% GC content were sufficient to direct self-assembly. A functional plasmid that exceeded 10 kb, which includes an in-frame fusion domain, was efficiently constructed from four PCR fragments in one step by our improved method.     

The rejoining of double-strand breaks in DNA by human cell extracts.

A double-strand DNA break was introduced at a specific site within the lacZ gene of plasmid pUC18 using one of several restriction enzymes, and the plasmid exposed to nuclear extracts from human cell lines. Physical rejoining of DNA was monitored by Southern analysis after gel separation, and the fidelity of rejoining by expression of the lacZ gene after bacterial transformation with the treated plasmid. Breaks at the SalI and EcoRI sites were rejoined by extracts to form circular monomers, but the efficiency of rejoining was much higher at the SalI site. Measurement of rejoining at several adjacent sites having different types of termini, consistently showed a range of efficiencies with 5' 4-base greater than 3' 4-base overhangs and 4-base greater than 2-base greater than no overhang. Similar efficiencies were found for nuclear extracts from transformed cell lines, both from a 'normal' individual and an ataxia-telangiectasia (A-T) patient, and from a non-transformed normal cell culture. In contrast at some sites, especially those with a low rejoin efficiency, the fidelity of rejoining was very much lower for the A-T extracts than for normal cell extracts. Mis-rejoining was, however, unrelated to rejoin efficiency at other sites, suggesting that factors such as the exact sequence at the break site on the molecule may also influence the fidelity of rejoining.     

Protection of DNA ends by telomeric 3' G-tail sequences

The extreme ends of eukaryotic chromosomes contain 3' extensions in the form of single-stranded G-rich repeats, referred to as telomeric 3' G-tails or overhangs. Increasing evidence has suggested that telomeric 3' G-tails can adopt a G-quadruplex conformation both in vitro and in vivo. However, the role of G-quadruplexes on the structure and function of telomeric 3' G-tails remains unclear. In the current study, we showed that the human telomeric 3' G-tail sequence protected the duplex DNA ends in cis from being recognized as double strand breaks. This protection is dependent on the G-quadruplex conformation of the 3' G-tail sequence. These results suggest that the ability of telomeric 3' G-tails to adopt the endprotecting G-quadruplex conformation may be one of the reasons for the existence of the evolutionarily conserved G-stretch motifs in telomeric DNA sequences.