Mapping of single-stranded regions in duplex DNA at the sequence level: single-strand-specific cytosine methylation in RNA polymerase-promoter complexes.

A method based on the differential rate of cytosine methylation in single- and double-stranded nucleic acids by dimethyl sulfate [Peattie, D.A. & Gilbert, W. (1980) Proc. Natl. Acad. Sci. USA 77, 4679-4682] has been developed for probing unpaired cytosines in DNA and DNA-protein complexes at the sequence level. Application of the method to the complexes between Escherichia coli RNA polymerase (EC 2.7.7.6) and three related promoters, lac UV5, trp, and a hybrid promoter tac resulting from the fusion of the two, reveals distinct differences in the way RNA polymerase unpairs DNA in these promoters. No single-stranded region is detectable in the complex with the trp promoter. For the lac UV5 promoter, the cytosines at positions -6, -4, -2, and -1 are in an unpaired region. The same cytosines in the tac promoter, which is homologous in sequence to lac UV5 in this region, are also found to be single stranded. For the pair of promoters lac UV5 and tac, the cytosine methylation reaction has also been used to demonstrate the steep temperature dependence of opening of base pairs by RNA polymerase. One striking feature is that the midpoint of this transition for the tac promoter is 3 degrees C lower than the corresponding value for lac UV5, even though the sequence of the unpaired region in the two promoters is identical.     

APOBEC3G hypermutates genomic DNA and inhibits Ty1 retrotransposition in yeast

Human cells harbor a variety of factors that function to block the proliferation of foreign nucleic acid. The APOBEC3G enzyme inhibits the replication of retroviruses by deaminating nascent retroviral cDNA cytosines to uracils, lesions that can result in lethal levels of hypermutation. Here, we demonstrate that APOBEC3G is capable of deaminating genomic cytosines in Saccharomyces cerevisiae. APOBEC3G expression caused a 20-fold increase in frequency of mutation to canavanine-resistance, which was further elevated in a uracil DNA glycosylase-deficient background. All APOBEC3G-induced base substitution mutations mapped to the nuclear CAN1 gene and were exclusively C/G --> T/A transition mutations within a 5'-CC consensus. The APOBEC3G preferred sites were found on both strands of the DNA duplex, but were otherwise located in hotspots nearly identical to those found previously in retroviral cDNA. This unique genetic system further enabled us to show that expression of APOBEC3G or its homolog APOBEC3F was able to inhibit the mobility of the retrotransposon Ty1 by a mechanism that involves the deamination of cDNA cytosines. Thus, these data expand the range of likely APOBEC3 targets to include nuclear DNA and endogenous retroelements, which have pathological and physiological implications, respectively. We postulate that the APOBEC3-dependent innate cellular defense constitutes a tightly regulated arm of a conserved mobile nucleic acid restriction mechanism that is poised to limit internal as well as external assaults.     

Variability of DNA methylation patterns during serial passage of human diploid fibroblasts

We have examined DNA methylation in diploid human fibroblasts, early and late in their replicative life-span. The extent of methylation of -C-C-G-G- was measured by comparison of fragment sizes after digestion with methylation-specific restriction enzyme Hpa II or Msp I, or both. Methylation of -C-C-G-G- sites in total DNA, occurring predominantly at internal (3′) cytosines, increased from 59% to 64% of sites in one cell strain at late passage, remained constant in another, and decreased in four other strains (54% to 48%, 58.5% to 48%, 55% to 51.5%, and 52% to 44.5%). Base composition analysis confirmed a substantial loss of total DNA 5-methylcytosine (mC) in one strain. Seven clonal isolates, examined at middle to late passage, ranged from 33% to 51% methylation of 3′ cytosines in -C-C-G-G- sites. Three discrete classes of highly repetitive DNA were found which contained Msp I sites at intervals of 45, 110, and 175 base pairs. These repeat families consistently had 70-80% of sites methylated at 3′ cytosines, in all clones and in all strains examined both at early and at late passage. Thus, altered methylation of repetitive sequences is unlikely to account for the variable -C-C-G-G- methylation observed in total DNA. When DNA from one fibroblast strain and from eight pure clones isolated from that parental culture was digested with Msp I or Hpa II followed by EcoRI and probed for γ-globin gene sequences, considerable interclonal and intraclonal heterogeneity was observed for methylation at four -C-C-G-G- sites in the γ-globin coding region of DNA. Therefore, the pattern of methylation in endogenous gene regions appears to undergo random drift during replication of diploid fibroblasts.     

Multiple rounds of adenovirus DNA synthesis in vitro

Adenovirus (Ad) type 2 DNA synthesis can be initiated in the presence of a soluble extract of uninfected HeLa cell nuclei, a 25-60% saturated ammonium sulfate fraction of infected cytoplasm and viral DNA covalently linked to a 5′-terminal protein (Ad DNA-prot). As the purification, from either the nuclei or cytoplasm, of factors active in DNA replication proceeded, various nonreplicative reactions which incorporate labeled deoxynucleotides were uncovered. In order to distinguish replicative from repair reactions, an assay was developed in which the Ad DNA-prot was digested with Xba I, all of the fragments so produced were used (without separation) in a replication reaction, and the products were assayed by electrophoresis on neutral agarose gels. In replicative reactions, most of the radioactivity was incorporated into the terminal fragments, with the internal fragments remaining unlabeled. Infected cytoplasm contains a “discrimination” function in addition to specific factors for Ad DNA replication. The discrimination factors inhibit the nonspecific nucleotide incorporation by uninfected HeLa nuclear extracts on Ad DNA-prot. The specific replicative incorporation into the terminal Ad DNA-prot fragments has also allowed an independent assay for reinitiation of progeny molecules synthesized in vitro. After the first round of replication, the 5′ strand of the progeny duplex from each end is labeled. These same labeled strands will be displaced during the second round of replication and appear in new bands which have been shown to be the single-strand equivalents of the terminal fragments. Thus, at least two rounds of Ad DNA synthesis can initiate at each terminus in vitro. The appearance of displaced single strands requires DNA replication because the addition of dideoxycytidine triphosphate after the first round of synthesis prevents the displacement reaction. Both the progeny single- and double-stranded DNA appear to be linked to protein.     

Sequence-specific recognition of cytosine C5 and adenine N6 DNA methyltransferases requires different deformations of DNA.

DNA methyltransferases modify specific cytosines and adenines within 2-6 bp recognition sequences. We used scanning force microscopy and gel shift analysis to show that M.HhaI, a cytosine C-5 DNA methyltransferase, causes only a 2 degree bend upon binding its recognition site. Our results are consistent with prior crystallographic analysis showing that the enzyme stabilizes an extrahelical base while leaving the DNA duplex otherwise unperturbed. In contrast, similar analysis of M.EcoRI, an adenine N6 DNA methyltransferase, shows an average bend angle of approximately 52 degrees. This distortion of DNA conformation by M.EcoRI is shown to be important for sequence-specific binding.     

Initiator RNA in Discontinuous Polyoma DNA Synthesis

During replication of polyoma DNA in isolated nuclei, RNA was found attached to the 5' ends of growing progeny strands. This RNA starts with either ATP or GTP and can be labeled at its 5' end with (32)P from beta-labeled nucleotides. Digestion of progeny strands with pancreatic DNase released (32)P-labeled RNA that, on gel electrophoresis, gave a distinct peak in the position expected for a decanucleotide. We believe that this short RNA is involved in the initiation of the discontinuous synthesis of DNA and propose the name "initiator RNA" for it. The covalent linkage of initiator RNA to 5' ends of growing DNA chains was substantiated by the finding that (32)P was transferred to ribonucleotides by alkaline hydrolysis of purified initiator RNA obtained by DNase digestion of polyoma progeny strands synthesized from [alpha-(32)P]dTTP. While initiator RNA was quite homogeneous in size, it had no unique base sequence since digestion with pancreatic RNase of initiator RNA labeled at its 5' end with (32)P released a variety of different [(32)P]oligonucleotides. The switch from RNA to DNA synthesis during strand elongation may thus depend on the size of initiator RNA rather than on a specific base sequence.     

Sequence information can be obtained from single DNA molecules

The completion of the human genome draft has taken several years and is only the beginning of a period in which large amounts of DNA and RNA sequence information will be required from many individuals and species. Conventional sequencing technology has limitations in cost, speed, and sensitivity, with the result that the demand for sequence information far outstrips current capacity. There have been several proposals to address these issues by developing the ability to sequence single DNA molecules, but none have been experimentally demonstrated. Here we report the use of DNA polymerase to obtain sequence information from single DNA molecules by using fluorescence microscopy. We monitored repeated incorporation of fluorescently labeled nucleotides into individual DNA strands with single base resolution, allowing the determination of sequence fingerprints up to 5 bp in length. These experiments show that one can study the activity of DNA polymerase at the single molecule level with single base resolution and a high degree of parallelization, thus providing the foundation for a practical single molecule sequencing technology.     

Base ratio, DNA content, and quinacrine-brightness of human chromosomes.

Human chromosomes were labeled with base-specific radioactive DNA precursors and examined autoradiographically to measure their DNA content and base ratio (percentage A-T base pairs). The requirement that incorporation of labeled bases be uniform during DNA synthesis was met by the use of inhibitors of de novo synthesis of DNA precursors. The genome was subdivided into 75 segments based on quinacrine banding, and the base ratio of each was calculated by a method that corrects for bias due to the scatter of grains about their source. Estimates of base ratio are shown to be sufficiently precise to detect variability among chromosomes and among segments within a chromosome. Analysis of these data and of measurements of the quinacrine fluorescence intensity of segments leads to the following conclusions. Base ratio is positively correlated with brightness, as predicted from independent in vitro studies. Larger chromosomes tend to have higher base ratios and to be brighter than smaller ones. The best prediction of the brightness of a segment must take into account not only its base ratio but also its DNA content. To explain these results, we suggest an evolutionary model in which chromosomes containing repeated sequences of A-T-rich DNA tend to grow by means of unequal sister chromatid and meiotic exchanges.     

Detection and characterization of RNA tumor virus-specific DNA in cells.

RNA tumor virus-specific DNA in cells can be detected by its capacity to 1) alter the reassociation kinetics of labeled double-stranded product of viral RNA-directed DNA polymerase; 2) anneal single-stranded DNA (cDNA) synthesized by viral polymerase; or 3) hybridize labeled viral 70S (genomic) RNA. Duplexes formed with these procedures can be analyzed for fidelity of base pairing, and the integration of viral DNA into the host genome can be established with a simple but stringent technique. We illustrate this methodology as applied to detection of Rous sarcoma virus (RSV)-specific DNA in XC cells and of mouse mammary tumor virus (MMTV)-specific DNA in murine and human tissues.     

Structure of nogalamycin bound to a DNA hexamer.

The anthracycline antibiotic nogalamycin, which binds to DNA, is composed of a planar aglycone substituted on each end to form an unusual dumbbell-shaped molecule. At one end nogalamycin contains an uncharged nogalose sugar and a methyl ester. At the other end nogalamycin contains a positively charged bicyclo amino sugar. We report the crystal structure of nogalamycin bound to the self-complementary DNA hexamer d(m5CGTsAm5CG). In this complex, the cytosines are methylated at the 5 position and the DNA contains a phosphorothioate linkage at the TpA step. Two nogalamycin molecules bind to the 6-base-pair fragment of double-helical DNA. The drug has threaded between the phosphodiester backbones with three aromatic rings intercalated within the DNA. In the major groove, the bicyclo amino sugar forms two direct hydrogen bonds to span a CG base pair and interacts indirectly with the next base pair of the duplex via a water-mediated hydrogen bond. In the minor groove, a carbonyl oxygen of nogalamycin forms a hydrogen bond directly to N2 of a guanine. The DNA base pairs are severely buckled by up to 26 degrees and are also distorted in directions perpendicular to the Watson-Crick hydrogen bonds. This complex illustrates the deformable nature of DNA.