Patterns of nucleotide misincorporations during enzymatic amplification and direct large-scale sequencing of ancient DNA.

Whereas evolutionary inferences derived from present-day DNA sequences are by necessity indirect, ancient DNA sequences provide a direct view of past genetic variants. However, base lesions that accumulate in DNA over time may cause nucleotide misincorporations when ancient DNA sequences are replicated. By repeated amplifications of mitochondrial DNA sequences from a large number of ancient wolf remains, we show that C/G-to-T/A transitions are the predominant type of such misincorporations. Using a massively parallel sequencing method that allows large numbers of single DNA strands to be sequenced, we show that modifications of C, as well as to a lesser extent of G, residues cause such misincorporations. Experiments where oligonucleotides containing modified bases are used as templates in amplification reactions suggest that both of these types of misincorporations can be caused by deamination of the template bases. New DNA sequencing methods in conjunction with knowledge of misincorporation processes have now, in principle, opened the way for the determination of complete genomes from organisms that became extinct during and after the last glaciation.

Metal-ion coordination by U6 small nuclear RNA contributes to catalysis in the spliceosome.

Introns are removed from nuclear messenger RNA precursors through two sequential phospho-transesterification reactions in a dynamic RNA-protein complex called the spliceosome. But whether splicing is catalysed by small nuclear RNAs in the spliceosome is unresolved. As the spliceosome is a metalloenzyme, it is important to determine whether snRNAs coordinate catalytic metals. Here we show that yeast U6 snRNA coordinates a metal ion that is required for the catalytic activity of the spliceosome. With Mg2+, U6 snRNA with a sulphur substitution for the pro-Rp or pro-Sp non-bridging phosphoryl oxygen of nucleotide U80 reconstitutes a fully assembled yet catalytically inactive spliceosome. Adding a thiophilic ion such as Mn2+ allows the first transesterification reaction to occur in the U6/sU80(Sp)- but not the U6/sU80(Rp)-reconstituted spliceosome. Mg2+ competitively inhibits the Mn2+-rescued reaction, indicating that the metal-binding site at U6/U80 exists in the wild-type spliceosome and that the site changes its metal requirement for activity in the Sp spliceosome. Thus, U6 snRNA contributes to pre-messenger RNA splicing through metal-ion coordination, which is consistent with RNA catalysis by the spliceosome.

Chiral copper-chelate complexes alter selectivities in metal affinity protein partitioning.

Proteins can be distinguished by exploiting complementarity between a histidine's microenvironment and a metal-chelate ligand in metal-affinity separations. The partitioning behavior of three myoglobins was investigated in aqueous two-phase polyethylene glycol-dextran systems containing polyethylene glycol derivatized with Cu(II) complexes of the L- and D-isomers of methionine and aspartate. TSK chromatographic supports derivatized with the methionine complexes were used to study retention of these proteins in metal-affinity chromatography. In the partitioning studies, the amino acid metal chelates exhibit selectivities for the myoglobins that are different from that of Cu(II)-iminodiacetate. Significant differences in selectivity based on the chiral nature of the amino acid complexes were also observed. The chromatographic selectivities of the chelating ligands exhibit little variation, however, suggesting that interactions occurring in solution but not on a surface play an important role in protein binding to the Cu(II)-amino acid-PEG complexes. In solution, the Cu(II)-amino acid complexes are sensitive probes of the microenvironments of surface histidines. The choice of the metal chelate affinity ligand offers a powerful means by which the selectivity of metal-affinity separations can be altered.

Metal affinity precipitation of proteins.

Proteins containing multiple surface-accessible histidine residues can be precipitated using small quantities of bis-copper chelates. The chelates serve to crosslink the proteins, presumably via the accessible histidines, leading to the formation of large, insoluble complexes. When excess copper chelate is used to carry out the precipitation, the resulting precipitate has a stoichiometry of 1:1 copper:accessible histidine. The precipitation is analogous to antibody-antigen precipitin reactions and can be described qualitatively using simple equilibrium theory developed for those systems. Human hemoglobin contains a large number of surface histidines and is efficiently precipitated by the copper salt CuSO4 as well as by bis-copper chelates. Sperm whale myoglobin contains many fewer surface histidines and is precipitated only by the bis-chelates. The effects of the number of accessible histidines on the protein, the chain length separating the two chelates, and the pH on the precipitation reaction have been investigated.