UV light-induced crosslinking of short DNA duplex strands: nucleotide sequence preferences and a prominent role of the duplex ends.

By various doses of UV light, we irradiated 32 short DNA duplexes having between 12 and 40 nucleotide residues in length, and monitored the induced crosslinks between the complementary DNA strands by denaturing polyacrylamide gel electrophoresis. The experiments revealed that the crosslinking was strongest with the alternating sequence of T and A and weaker with the alternating sequence of T and G (C and A in the complementary strand). On the other hand, GC blocks of any sequence provided undetectable amounts of interstrand crosslinks even at the highest doses of UV irradiation. The amount of crosslinked strands logarithmically increased with the UV dose but it did not depend on the oligonucleotide concentration, ionic strength, divalent magnesium or manganese cations and pH at least within the examined regions of the experimental conditions, unless the oligonucleotide denatured or isomerized into a unimolecular foldback. The extent of crosslinking also did not depend on the (dT-dA)n duplex length to indicate that the crosslink was predominantly localized at a specific duplex locus. Experiments with (dT-dA)8 "mutants" in which AT pairs were systematically replaced by GC pairs at various molecule positions, revealed that the crosslinking predominantly occurred at the oligo(dT-dA) duplex ends. The crosslinking is a direct method to detect duplexes of DNA, which is here, for example, demonstrated with the heteroduplex of (dT-dA)12 and (dT-dA)16.

Biased distribution of adenine and thymine in gene nucleotide sequences.

We analyzed occurrences of bases in 20,352 introns, exons of 25,574 protein-coding genes, and among the three codon positions in the protein-coding sequences. The nucleotide sequences originated from the whole spectrum of organisms from bacteria to primates. The analysis revealed the following: (1) In most exons, adenine dominates over thymine. In other words, adenine and thymine are distributed in an asymmetric way between the exon and the complementary strand, and the coding sequence is mostly located in the adenine-rich strand. (2) Thymine dominates over adenine not only in the strand complementary to the exon but also in introns. (3) A general bias is further revealed in the distribution of adenine and thymine among the three codon positions in the exons, where adenine dominates over thymine in the second and mainly the first codon position while the reverse holds in the third codon position. The product (A1/T1)x(A2/T2)x(T3/A3) is smaller than one in only a few analyzed genes.

Conformational isomerizations of poly(dA-dT) are dramatically influenced by a substitution of a minor amount of adenine by purine or amino2purine.

We have synthesized poly(dA,dPu-dT) and poly(dA,n2dPu-dT) containing, respectively, 5.7% of purine and 7.4% of amino2purine in place of adenine to demonstrate that these apparently negligible perturbations of the primary structure have dramatic consequences for the polynucleotide conformational isomerizations. The replacement of adenine by amino2purine, preserving the number of hydrogen bonds between the complementary bases, has a stronger effect on the polynucleotide conformational isomerizations than the replacement with purine that is bound only by a single hydrogen bond to thymine. Nevertheless, poly(dA,dPu-dT) forms a more thermostable duplex than poly(dA,n2dPu-dT). Furthermore the few amino2purines in poly(dA,n2dPu-dT) inhibit its isomerization into X-DNA, stabilize but modify A-DNA and stabilize Z-DNA. Kinetics of the B-Z transition of poly(dA,n2dPu-dT) is fast to indicate that the amino groups in the double helix minor groove substantially decrease the kinetic barrier between B- and Z-DNA. On the other hand, the replacement of adenine by purine destabilizes both Z-DNA and A-DNA, and the destabilization of X-DNA is weaker than with amino2purine. A-form and B-form perhaps coexist in poly(dA,dPu-dT) at high concentrations of ethanol.