Instability of simple sequence DNA in Saccharomyces cerevisiae.

All eukaryotic genomes thus far examined contain simple sequence repeats. A particularly common simple sequence in many organisms (including humans) consists of tracts of alternating GT residues on one strand. Allelic poly(GT) tracts are often of different lengths in different individuals, indicating that they are likely to be unstable. We examined the instability of poly(GT) and poly(G) tracts in the yeast Saccharomyces cerevisiae. We found that these tracts were dramatically unstable, altering length at a minimal rate of 10(-4) events per division. Most of the changes involved one or two repeat unit additions or deletions, although one alteration involved an interaction with the yeast telomeres.     

Influence of DNA sequence on the nature of mispairing during DNA synthesis

A series of synthetic oligonucleotide primers, annealed at various positions along the lacZ-alpha region of bacteriophage M13mp9 template, were elongated by purified DNA polymerases in the presence of only 3 of the 4 deoxynucleoside triphosphates to achieve misincorporation at a total of 49 different positions along the template. The newly synthesized strands (containing misincorporated bases) were isolated and sequenced to determine the identity of misincorporated deoxynucleoside monophosphates. The results indicate that the kind of mispairing that occurs during DNA synthesis is greatly influenced by the nucleotide sequence of the template. Transition-type base substitutions predominated overall, but at many template positions, transversion-type base substitutions occurred, most commonly via A.A mispairing. The results of parallel determinations made with Escherichia coli DNA polymerase I ("large fragment" form) and DNA polymerase of Maloney murine leukemia virus indicated that, overall, the identity of polymerase had only a small effect on the kind of misincorporation that occurred at different positions along the template. However, at certain template positions, the nature of mispairing during DNA synthesis was reproducibly affected by differing polymerase active-site environment.     

Identification of the initiation sequence for viral-strand DNA synthesis of wheat dwarf virus.

The intergenic region of the circular single-stranded DNA genome of geminiviruses contains a sequence potentially able to fold into a stem-loop structure. This sequence has been reported to be involved in viral replication by serving as the origin for rolling-circle replication. However, in wheat dwarf virus (WDV) a deletion of 128 bp, removing this sequence, surprisingly does not prevent de novo viral DNA synthesis, but instead abrogates the processing of replicative intermediates into monomeric genomes. This deletion mutant permitted us to study the initiation of viral-strand DNA synthesis independently from its termination and also to identify the sequence within which rolling-circle DNA replication of WDV begins. We have mapped the initiation site of replication to a pentanucleotide, TACCC, a sequence that occurs twice in the large intergenic region of WDV: it is found in the right half of the stem-loop sequence and again 170 bases upstream where it is part of a 15 nucleotide sequence highly homologous to the right half of the stem-loop sequence. Here we show that viral-strand DNA synthesis efficiently initiates at both sequences.     

Chemical synthesis of tridecanucleoside dodecaphosphate sequence of SV40 DNA

The preparation, by the phosphotriester approach, of d[C-T-A-T-T-C-C-A-G-A-A-G-T] from one tetranucleoside triphosphate and three trinucleoside diphosphate blocks is described. The use of the o-dibromomethylbenzoyl (DBMB) protecting group in oligodeoxyribonucleotide synthesis is described for the first time. Internucleotide linkages are protected by o-chlorophenyl groups which are finally removed by treatment with the N1, N1, N3, N3-tetramethylguanidinium salt of syn-4-nitrobenzaldoxime. The first phosphorylation step (leading to phosphodiester intermediates) is carried out by treatment with o-chlorophenyl phosphorodi-(1,2,4-triazolide) followed by treatment with water and triethylamine. 1-Mesitylenesulphonyl-3-nitro-1,2,4-triazole (MSNT) is used as the activating agent in the second phosphorylation step in which 5'-protected mono- and di-nucleotides are condensed with nucleoside building blocks containing unprotected 3'-hydroxy functions.     

Molecular dynamics of spermine-DNA interactions: sequence specificity and DNA bending for a simple ligand.

We used molecular dynamics to model interactions between the physiologically important polyamine spermine and two B-DNA oligomers, the homopolymer (dG)10-(dC)10 and the heteropolymer (dGdC)5-(dGdC)5. Water and counterions were included in the simulation. Starting coordinates for spermine-DNA complexes were structures obtained by molecular mechanics modeling of spermine with the two oligomers; in these models, spermine binding induced a bend in the heteropolymer but not in the homopolymer. During approximately 40 psec of molecular dynamics simulation, spermine moves away from the floor of the major groove and interacts nospecifically with d(G)10-d(C)10. In contrast, a spermine-induced bend in the helix of (dGdC)5-(dGdC)5 is maintained throughout the simulation and spermine remains closely associated with the major groove. These results provide further evidence that the binding of spermine to nucleic acids can be sequence specific and that bending of alternating purine-pyrimidine sequences may be a physiologically important result of spermine binding.     

A theoretical model for template-free synthesis of long DNA sequence

This theoretical scheme is intended to formulate a potential method for high fidelity synthesis of Nucleic Acid molecules towards a few thousand bases using an enzyme system. Terminal Deoxyribonucleotidyl Transferase, which adds a nucleotide to the 3′OH end of a Nucleic Acid molecule, may be used in combination with a controlled method for nucleotide addition and degradation, to synthesize a predefined Nucleic Acid sequence. A pH control system is suggested to regulate the sequential activity switching of different enzymes in the synthetic scheme. Current practice of synthetic biology is cumbersome, expensive and often error prone owing to the dependence on the ligation of short oligonucleotides to fabricate functional genetic parts. The projected scheme is likely to render synthetic genomics appreciably convenient and economic by providing longer DNA molecules to start with.     

Deletion Mutations Caused by DNA Strand Slippage in Acinetobacter baylyi

Short nucleotide sequence repetitions in DNA can provide selective benefits and also can be a source of genetic instability arising from deletions guided by pairing between misaligned strands. These findings raise the question of how the frequency of deletion mutations is influenced by the length of sequence repetitions and by the distance between them. An experimental approach to this question was presented by the heat-sensitive phenotype conferred by pcaG1102, a 30-bp deletion in one of the structural genes for Acinetobacter baylyi protocatechuate 3,4-dioxygenase, which is required for growth with quinate. The original pcaG1102 deletion appears to have been guided by pairing between slipped DNA strands from nearby repeated sequences in wild-type pcaG. Placement of an in-phase termination codon between the repeated sequences in pcaG prevents growth with quinate and permits selection of sequence-guided deletions that excise the codon and permit quinate to be used as a growth substrate at room temperature. Natural transformation facilitated introduction of 68 different variants of the wild-type repeat structure within pcaG into the A. baylyi chromosome, and the frequency of deletion between the repetitions was determined with a novel method, precision plating. The deletion frequency increases with repeat length, decreases with the distance between repeats, and requires a minimum amount of similarity to occur at measurable rates. Deletions occurred in a recA-deficient background. Their frequency was unaffected by deficiencies in mutS and was increased by inactivation of recG.     

Nucleotide sequence of the DNA packaging and capsid synthesis genes of bacteriophage P2.

Overlapping DNA fragments containing the DNA packaging and capsid synthesis gene region of bacteriophage P2 were cloned and sequenced. In this report we present the complete nucleotide sequence of this 6550 bp region. Each of six open reading frames found in the interval was assigned to one of the essential genes (Q, P, O, N, M and L) by correlating genetic, physical and mutational data with DNA and protein sequence information. Polypeptides predicted were: a capsid completion protein, gpL; the major capsid precursor, gpN; the presumed capsid scaffolding protein; gpO; the ATPase and proposed endonuclease subunits of terminase, gpP and gpM, respectively; and a candidate for the portal protein, gpQ. These gene and protein sequences exhibited no homology to analogous genes or proteins of other bacteriophages. Expression of gene Q in E. coli from a plasmid caused production of a Mr 39,000 Da protein that restored Qam34 growth. This sequence analysis found only genes previously known from analysis of conditional-lethal mutations. No new capsid genes were found.     

Telomeric DNA sequence and structure following de novo telomere synthesis in Euplotes crassus.

To learn more about the mechanism of de novo telomere synthesis, we have characterized the sequence and structure of newly synthesized telomeres from Euplotes crassus. E. crassus is a particularly useful organism for studying telomere synthesis because millions of telomeres are made in each cell at a well-defined time during the sexual stage of the life cycle. These newly synthesized telomeres are approximately 50 bp longer than mature macronuclear telomeres. We have investigated the structure of the newly synthesized telomeres and have found that they are much more heterogeneous in length than mature telomeres. Most of the heterogeneity is present on the G-rich strand, indicating that the length of this strand is rather loosely controlled. In contrast, the length of the C-rich strand is much less variable, suggesting that synthesis of this strand is the more precisely regulated step in telomere addition. The G-rich strand exhibits variability both in the total number of G4T4 repeats and in the identity of the terminal nucleotide. In most cases, the G-rich strnd extends beyond the C-rich strand to leave a 3' overhang. While the size of this overhang is variable, the median length is 10 nucleotides. This research provides the first detailed picture of a newly synthesized telomere and has allowed us to formulate a model to describe the various steps involved in de novo telomere synthesis.     

A simple method for global sequence comparison.

A simple method of sequence comparison, based on a correlation analysis of oligonucleotide frequency distributions, is here shown to be a reliable test of overall sequence similarity. The method does not involve sequence alignment procedures and permits the rapid screening of large amounts of sequence data. It identifies those sequences which deserve more careful analysis of sequence similarity at the level of resolution of the single nucleotide. It uses observed quantities only and does not involve the adoption of any theoretical model.     

Displacement synthesis of globin complementary DNA: evidence for sequence amplification

We have examined a cDNA displacement synthesis procedure in which the extent of precursor incorporation and the unusual kinetics of displacement synthesis suggest a unique replicative form of DNA and the occurrence of multiple rounds of displacement synthesis, leading to amplification of mRNA sequences. Globin double-stranded DNA containing a hairpin loop was extended by the addition of a homopolymer to the 3' end. This was followed by displacement synthesis with the Klenow fragment of DNA polymerase I that was primed by an oligonucleotide hybridized to the homopolymer. Thus, the hairpin cDNA was copied to form an open duplex with an inverted repetition of globin sequences. These molecules can then serve as templates for additional synthesis which would be primed from oligomers bound the homopolymer. Globin cDNA sequences appear to be amplified 10-fold or more by this procedure. Globin cDNA obtained by displacement synthesis was similar in size to the original template. However, displaced molecules associate to the extent that they are not readily resolved by electrophoresis or sedimentation under nondenaturing conditions. Restriction endonuclease digests of 32P-labeled displaced strands gave fragment patterns similar to rabbit globin cDNA hairpin molecules. S1 nuclease studies demonstrated that displaced complexes and replication intermediates are partially single stranded, which might account for their aggregation properties.