A novel double lesion in X-irradiated DNA consists of a strand break and a base modification.

A single free radical-initiating event can produce a pair of base lesions in DNA oligomers exposed to ionizing radiation. Whereas double base lesions have been observed previously, the present study shows that double lesions may sometimes consist of a base lesion and an associated strand break. The mechanism for the formation of double lesions is discussed. A redox process is postulated in which guanine is the source of the electron. It is suggested that double lesions may be formed in DNA either on adjacent nucleotides or, alternatively, on nucleotides separated by one, two or possibly more intervening nucleotides. It is hypothesized that intramolecular electron transfer facilitates the formation of double lesions on nonadjacent nucleotides.     

The interaction of p53 with 3'-terminal mismatched DNA

The diversity of p53 functions involves its interaction with sequence-specific, non-sequence-specific and various damaged sites in DNA. The preferential excision of misincorporated over correct nucleotides by the 3′→5′ exonuclease activity of p53 provides a molecular basis for p53 involvement in the correction of the DNA replication errors. However, p53 exhibits variations in its comparative efficiency to excise different 3'-terminal mismatched nucleotides. To determine the importance of the binding capacity of the protein to various 3'-terminal damaged sites, we have examined the interaction of p53 with linear dsDNAs containing various 3'-terminal mismatches, employing a gel retardation assay. The data demonstrate the intrinsic 3'-terminal mismatched DNA binding capacity of p53. Since p53 binds directly to various 3'-terminal purine:pyrimidine and purine:purine mispairs to an equal extent, p53 can be considered as a general 3'-mismatched DNA binding protein. Apparently, 3'-terminal mismatched bases are structural element to which p53 can bind, that extends the spectrum of damage sites to which p53 may respond. The formation of the p53-mismatched DNA complex is independent of the sequence context. Thus, the dissimilarities in mispair excision efficiency are probably due to an inherent property of the p53 in excision of 3'-mismatched nucleotides by a bound protein. The results establish a framework for understanding the mechanism of cooperative interaction between p53 and exonuclease-deficient DNA polymerase (e.g. HIV-1 RT). Within the context of error-correction events, p53 by recognition and excision of 3'-mismatched nucleotides from DNA, may be involved in DNA repair, thus increasing the accuracy of DNA synthesis by DNA polymerases.     

Adeno-associated virus type 2 DNA replication in vivo: mutation analyses of the D sequence in viral inverted terminal repeats.

The adeno-associated virus type 2 (AAV) genome contains inverted terminal repeats (ITRs) of 145 nucleotides. The terminal 125 nucleotides of each ITR form palindromic hairpin (HP) structures that serve as primers for AAV DNA replication. These HP structures also play an important role in integration as well as rescue of the proviral genome from latently infected cells or from recombinant AAV plasmids. Each ITR also contains a stretch of 20 nucleotides, designated the D sequence, that is not involved in HP structure formation. We have recently shown that the D sequence plays a crucial role in high-efficiency rescue, selective replication, and encapsidation of the AAV genome and that a host cell protein, designated the D sequence-binding protein (D-BP), specifically interacts with this sequence (X.-S. Wang, S. Ponnazhagan, and A. Srivastava, J. Virol. 70:1668-1677, 1996). We have now performed mutational analyses of the D sequences to evaluate their precise role in viral DNA rescue, replication, and packaging. We report here that 10 nucleotides proximal to the HP structure in each of the D sequences are necessary and sufficient to mediate high-efficiency rescue, replication, and encapsidation of the viral genome in vivo. In in vitro studies, the same 10 nucleotides were found to be required for specific interaction with D-BP, but viral Rep protein-mediated cleavage at the functional terminal resolution site is independent of these sequences. These data suggest that AAV replication and terminal resolution functions can be uncoupled and that the lack of efficient replication of AAV DNA may not be a consequence of impaired resolution of the viral ITRs. These studies further illustrate that the D sequence-D-BP interaction plays an important role in the AAV life cycle and indicate that it may be possible to develop the next generation of AAV vectors capable of encapsidating larger pieces of DNA.     

A re-examination of the crystal structure of A-DNA using fiber diffraction data

Classical A-DNA helices with h = 0.25 nm may represent the greatest mass per unit length attainable by polynucleotide duplexes. The X-ray diffraction pattern from polycrystalline and well-oriented fibers of calf thymus DNA in its A-form has been carefully re-examined. Indexing on the basis of a C-face-centered monoclinic unit cell of dimensions a = 2.170 nm, b = 3.990 nm, c = 2.803 nm and beta = 96.82 degrees is superior to alternatives that have been proposed. Two right-handed. Watson-Crick base-paired, helical DNA chains with 2 X 11 nucleotides per 2.803 nm pitch, each carrying C3'-endo furanose rings, pass through the unit cell. The crystallography requires the two chains in the duplex to be antiparallel and conformationally identical but the 11 nucleotides in each pitch may be distinct. However, a secondary structure with a mononucleotide asymmetric unit provides as good an X-ray agreement as one with 11 distinct nucleotides. This relative lack of variability is quite different from what is observed in fibrous B-DNAs.     

Statistical analysis of nucleotide runs in coding and noncoding DNA sequences

A statistical analysis of the occurrence of particular nucleotide runs in DNA sequences of different species has been carried out. There are considerable differences of run distributions in DNA sequences of procaryotes, invertebrates and vertebrates. There is an abundance of short runs (1-2 nucleotides long) in the coding sequences and there is a deficiency of such runs in the noncoding regions. However, some interesting exceptions from this rule exist for the run distribution of adenine in procaryotes and for the arrangement of purine-pyrimidine runs in eucaryotes. The similarity in the distributions of such runs in the coding and noncoding regions may be due to some structural features of the DNA molecule as a whole. Runs of guanine (or cytosine) of three to six nucleotides occur predominantly in noncoding DNA regions in eucaryotes, especially in vertebrates.     

Junk DNA Contribution to Evolutionary Capacitance Can Drive Species Dynamics

Junk DNA is still an enigmatic concept. Although junk DNA composition, abundance, and functionality are still contentious, its contribution to biological evolution is less questionable. Recently, I proposed that sexually restricted chromosomes such as Y and W, highly enriched in junk DNA elements, act as genomic tuning knobs indirectly causing a genome-wide increase in gene expression heterogeneity that boosts heterogametic individuals ability to endure environmental challenges and evolutionary capacitance, i.e., the store of genetic variation with no phenotypic effect. Sexually restricted chromosomes-based evolutionary capacitance might importantly contribute to metazoan sexual dimorphisms for dispersal and sex-biased gene expression dynamics. In this Synthesis, I hypothesize that large differences between species in the overall amount of junk DNA within their genomes also promote differences in junk DNA-based evolutionary capacitance that might be reflected in differences for dispersal and genetic diversification. I hypothesize that populations for species with junk DNA-impoverished genomes would show an enhanced ability to genetically diversify leading to a faster speciation rate even in the absence of geographic isolation when compared with populations for species with junk DNA-enriched genomes. To support junk DNA variation-based evolutionary capacitance effect on species genetic diversification, I analyzed the covariation of genome size as proxy for the overall amount of junk DNA in the genome and several genetic diversification measures obtained from interspecific crosses for the Drosophilidae family. The potential effect of junk DNA variation-based evolutionary capacitance for other elements of species dynamics such as extinction or the participation in grouped ecological structures is also briefly discussed.     

A DNA primase that copurifies with the major DNA polymerase from the yeast Saccharomyces cerevisiae

Biochemical fractionation of the yeast Saccharomyces cerevisiae has revealed a novel DNA primase activity that copurifies with the major DNA polymerase activity. In the presence of RNA precursors and single-stranded DNA (poly(dT), M13), the DNA primase synthesizes discrete length oligoribonucleotides (apparent length, 8-12 nucleotides) as well as longer RNA chains that appear to be multiples of a modal length of 11-12 nucleotides. When DNA precursors are also present, the oligoribonucleotides are utilized by the accompanying DNA polymerase as primers for DNA synthesis. Copurification of these two enzymatic activities suggests their association in a physical complex which may function in the synthesis of Okazaki fragments at chromosomal replication forks.     

Efficiency and fidelity of human DNA polymerases λ and β during gap-filling DNA synthesis

The base excision repair (BER) pathway coordinates the replacement of 1-10 nucleotides at sites of single-base lesions. This process generates DNA substrates with various gap sizes which can alter the catalytic efficiency and fidelity of a DNA polymerase during gap-filling DNA synthesis. Here, we quantitatively determined the substrate specificity and base substitution fidelity of human DNA polymerase λ (Pol λ), an enzyme proposed to support the known BER DNA polymerase β (Pol β), as it filled 1-10-nucleotide gaps at 1-nucleotide intervals. Pol λ incorporated a correct nucleotide with relatively high efficiency until the gap size exceeded 9 nucleotides. Unlike Pol λ, Pol β did not have an absolute threshold on gap size as the catalytic efficiency for a correct dNTP gradually decreased as the gap size increased from 2 to 10 nucleotides and then recovered for non-gapped DNA. Surprisingly, an increase in gap size resulted in lower polymerase fidelity for Pol λ, and this downregulation of fidelity was controlled by its non-enzymatic N-terminal domains. Overall, Pol λ was up to 160-fold more error-prone than Pol β, thereby suggesting Pol λ would be more mutagenic during long gap-filling DNA synthesis. In addition, dCTP was the preferred misincorporation for Pol λ and its N-terminal domain truncation mutants. This nucleotide preference was shown to be dependent upon the identity of the adjacent 5'-template base. Our results suggested that both Pol λ and Pol β would catalyze nucleotide incorporation with the highest combination of efficiency and accuracy when the DNA substrate contains a single-nucleotide gap. Thus, Pol λ, like Pol β, is better suited to catalyze gap-filling DNA synthesis during short-patch BER in vivo, although, Pol λ may play a role in long-patch BER.     

DNA repair excision nuclease attacks undamaged DNA. A potential source of spontaneous mutations

Nucleotide excision repair is a general repair system that eliminates many dissimilar lesions from DNA. In an effort to understand substrate determinants of this repair system, we tested DNAs with minor backbone modifications using the ultrasensitive excision assay. We found that a phosphorothioate and a methylphosphonate were excised with low efficiency. Surprisingly, we also found that fragments of 23-28 nucleotides and of 12-13 nucleotides characteristic of human and Escherichia coli excision repair, respectively, were removed from undamaged DNA at a significant rate. Considering the relative abundance of undamaged DNA in comparison to damaged DNA in the course of the life of an organism, we conclude that, in general, excision from and resynthesis of undamaged DNA may exceed the excision and resynthesis caused by DNA damage. As resynthesis is invariably associated with mutations, we propose that gratuitous repair may be an important source of spontaneous mutations.     

Mechanism of inhibition of bacteriophage T7 DNA synthesis in Escherichia coli B cells infected by alkylated bacteriophage T7

Quantitative analysis of DNA replication, in E. coli B cells infected by methyl methanesulfonate-treated bacteriophage T7, showed that production of phage DNA was delayed and decreased. The cause of the delay appeared to be a delay in host-DNA breakdown, the process which provides nucleotides for phage-DNA synthesis. In addition, reutilisation of host-derived nucleotides was impaired. These observations can be accounted for by a model in which methyl groups on phage DNA slow down DNA injection and also reduce the replicational template activity of the DNA once it has entered the cell. Repair of alkylated phage DNA may be required not only for replication but also for normal injection of DNA.     

Pancreatic DNAase cleavage sites in nuclei

The DNA of nuclei is cleaved by a variety of nucleases in such a way that the cuts on a given strand are always separated by an integral multiple of 10 nucleotides. However, the spacing between cutting sites on opposite strands is not known for any nuclease. In this paper, we describe the determination of the spacing, or stagger, between cuts on opposite strands produced by the action of pancreatic DNAase (DNAase I) on nuclei. When nuclei are digested with DNAase I and the resultant DNA is analyzed by gel electrophoresis without prior denaturation, a complex pattern of bands is observed. A method which gives better than 90% recovery of DNA from polyacrylamide gels was used to isolate the individual fractions corresponding to these bands. The structure of the fractions was then determined using single-strand-specific nuclease to digest single-stranded "tails" and using DNA polymerases to extend recessed 3'-OH termini of partially duplex regions. Our results show that each component consists of a double-stranded region terminating in single-stranded tails at both ends. Although both chains of every duplex are 10-n nucleotides long (n integer), the chains are never completely paired. The experiments with DNA polymerase show an abundance of structures in which the 3'-OH termini of these duplexes are recessed by 8 nucleotides, and by inference, there must be structures with 5'-P termini recessed by 2 or 12 nucleotides. Thus DNAase I acts on nuclei to produce DNA with staggered cuts on opposite strands, separated by (10-n + 8) and (10-n + 2) base pairs (with 5'-P and 3'-OH termini extending, respectively). Two classes of models of DNA folding in the nucleosome have been proposed by other investigators to account for the presence of DNAase I cleavage sites at 10-n intervals along each DNA chain. One class of models leads to the prediction that cuts should either be unstaggered or separated by 10 nucleotides, while the other class is consistent with staggers of 6 and 4 nucleotides. Neither prediction is verified by our data; however, all these models may be made consistent with the results by assuming that the enzyme's site of recognition on nucleosomal DNA is not the same as its site of cleavage.     

Same-Sex Twin Pair Phenotypic Correlations are Consistent with Human <Emphasis Type="Italic">Y</Emphasis> Chromosome Promoting Phenotypic Heterogeneity

Junk DNA has been long appreciated as an evolutionary facilitator because it can participate in the causation of genetic variation such as chromosome rearrangements and can be exapted into coding or regulatory elements. Recently, it has been proposed that junk DNA variation within natural populations indirectly causes a phenotypic heterogeneity that subsequently promotes genetic capacitance, i.e., the random fluctuation of genetic variation. Junk DNA role as capacitor might drive population traits such as sexual dimorphism, spatiotemporal dynamics, or genetic diversification leading into speciation. Whether the human species also showed junk DNA-based capacitance manifested as a junk DNA-dependent phenotypic heterogeneity that contributed to the etiology and expression of diseases or the evolutionary history of human populations is intriguing. Because the human Y chromosome is highly enriched in junk DNA, humans are sexually dimorphic for the genomic content in junk DNA. Thus, it would be expected that junk DNA-based capacitance in humans were manifested as a sexual dimorphism for phenotypic heterogeneity. Here, I gather supporting evidence for the existence of a sexual dimorphism for putative junk DNA-based phenotypic heterogeneity by analyzing same-sex twin pairs phenotypic concordance.     

Nucleic acids from the host bacterium as a major source of nucleotides for three marine bacteriophages

Abstract The incorporation of ³²P-phosphorus into marine bacteriophage nucleic acid was studied in culture experiments to investigate the source of nucleotides used by the phage. We consistently found that the ³²P-specific activity in the phage genome increased during the 11 h incubation and was low relative to the specific activity in the medium, averaging 21% (±SD 5.9) for the three phage isolates. This was in accordance with a mathematical model where most of the nucleotides for phage DNA synthesis were derived from the host cell nucleic acid rather than de novo synthesis. We propose that this metabolic strategy may be common among marine phages, as an adaptation to a nutrient poor environment. Consequently, the contribution of free DNA to the dissolved fraction through phage lysis of bacteria, may be less that previously thought. Also during radiolabelling of bacteriophages in natural water samples, isotope dilution may be dependent on the specific growth rate of the bacterial host.     

DNA sequencing with direct blotting electrophoresis.

A method for transferring the DNA molecules of sequencing reaction mixtures onto an immobilizing matrix during electrophoresis has been developed. A blotting membrane moves with constant speed across the end of a very short, denaturing gel and collects the molecules according to size. A constant distance between bands for molecules differing in length by one nucleotide is obtained over a large range (approximately 600 nucleotides with a 5% gel), simplifying the determination of DNA sequences considerably. Reliable sequences of 500 nucleotides can be read and sequence features up to greater than 1000 nucleotides are revealed in a single experiment. The sequencing of a potential Z-DNA-forming fragment from Escherichia coli DNA is given as an example and possible further developments are discussed.     

Characteristics of nucleotide blocks in coding and non-coding DNA sequences from different organisms

A statistical analysis of occurrence of particular nucleotide runs (1 divided by 10 nucleotides long) in DNA sequences of different species has been carried out. There are considerable differences in run distributions in DNA sequences of prokaryotes, invertebrates and vertebrates. Distribution of various types of runs has been found to be different in coding and non-coding sequences. There is an abundance of short runs 1 divided by 2 nucleotides long in coding sequences, and there is a deficiency of such runs in the non-coding regions. However, some interesting exceptions from this rule exist: for run distribution of adenine in prokaryotes and for distribution of purine-pyrimidine runs in eukaryotes. This may be stipulated by the fact that the distribution of runs are predetermined by structural peculiarities of the entire DNA molecule. Runs of guanine or cytosine of three to six nucleotides long occur predominantly in the non-coding DNA regions in eukaryotes, especially in vertebrates.     

Comparison of filler DNA at immune, nonimmune, and oncogenic rearrangements suggests multiple mechanisms of formation.

Extra nucleotides (termed filler DNA) are commonly found at the junctions of genetic rearrangements in mammalian cells. The filler DNA at immune system rearrangements, which are called N regions, are generated at VDJ joints primarily by terminal deoxynucleotidyl transferase. However, the origin of filler DNA at genetic rearrangements in nonlymphoid cells is uncertain. In an analysis of more than 200 junctions that arose by circularization of transfected linear DNA (D. B. Roth and J. H. Wilson, Mol. Cell. Biol. 6:4295-4304, 1986), we found 18 junctions with extra nucleotides exactly at the point of circularization. Analysis of these 18 junctions indicated that nonlymphoid cells could add extra nucleotides to the ends of duplex DNA. The characteristics of the extra nucleotides at these junctions and at 31 other rearrangement junctions from nonlymphoid cells were quite similar, suggesting that many genetic rearrangements may pass through a stage with free DNA ends. A comparison of the filler DNA at these 49 nonimmune system rearrangements with 97 N regions derived from immune system rearrangements suggested that lymphoid and nonlymphoid cells use different mechanisms for insertion of filler DNA, as expected from the absence of detectable terminal deoxynucleotidyl transferase in cells from nonlymphoid tissues. The filler DNAs at a smaller group of 22 translocations associated with cancer had features in common with both immune and nonimmune system rearrangements and therefore may represent a mixture of these two processes. Mechanisms that might account for the presence of filler DNA in nonlymphoid cells are discussed.     

Regularities of the content and organization of pyrimidine oligonucleotide sequences in insect DNA

Certain regularities in content and organization of pyrimidine oligonucleotide sequences of DNA from 15 insect species belonging to 4 orders were studied. The degree of nucleotide clusterization in insect DNA was found to be species-specific, being the highest in Hymenoptera and lowest in Lepidoptera; the Blattodea and Coleoptera occupy an intermediate position by this index between them. The changes in the DNA cluster structure during the evolution of insect species are not of vector type; the degree of clusterization of DNA nucleotide is either increased (Hymenoptera) or decreased (Lepidoptera as compared with Blattodea). In the DNA oligonucleotide fractions containing both pyrimidine nucleotides the percentage content of thymidyl nucleotides is much higher than that of cytidyl nucleotides, the thymine content being increased with the lengthening of oligopyrimidine clusters. The insect species with a higher degree of clusterization of DNA pyrimidine nucleotides contain more thymidyl nucleotide residues. These results agree well with the hypothesis suggesting that during the evolution of large taxons the accumulation of long pyrimidine sequences in animal DNA is accompanied by an increase of thymidyl nucleotide content in them. This can largely be due to the increase of matrix resistance during the evolution and is biologically significant for animals of any taxons, including insects.     

Dissection of RNA-primed DNA synthesis catalyzed by gene 4 protein and DNA polymerase of bacteriophage T7. Coupling of RNA primer and DNA synthesis

Gene 4 protein and DNA polymerase of bacteriophage T7 catalyze RNA-primed DNA synthesis on single-stranded DNA templates. T7 DNA polymerase exhibits an affinity for both gene 4 protein and single-stranded DNA, and gene 4 protein binds stably to single-stranded DNA in the presence of dTTP (Nakai, H. and Richardson, C. C. (1986) J. Biol. Chem. 261, 15208-15216). Gene 4 protein-T7 DNA polymerase-template complexes may be formed in both the presence and absence of nucleoside 5'-triphosphates. The protein-template complexes may be isolated free of unbound proteins and nucleotides by gel filtration and will catalyze RNA-primed DNA synthesis in the presence of ATP, CTP, and the four deoxynucleoside 5'-triphosphates. RNA-primed DNA synthesis may be dissected into separate reactions for primer synthesis and DNA synthesis. Upon incubation of gene 4 protein with single-stranded DNA, ATP, and CTP, a primer-template complex is formed; it is likely that gene 4 protein mediates stable binding of the oligonucleotide to the template. The complex, purified free of unbound proteins and nucleotides, supports DNA synthesis upon addition of DNA polymerase and deoxynucleoside 5'-triphosphates. Association of primers with the template is increased by the presence of dTTP or DNA polymerase during primer synthesis. DNA synthesis supported by primer-template complexes initiates predominantly at gene 4 recognition sequences, indicating that primers are bound to the template at these sites.