Replication of non-hydrogen bonded bases by DNA polymerases: A mechanism for steric matching

Recent experiments have presented evidence that Watson–Crick hydrogen bonds in a base pair are not absolute requirements for efficient synthesis of that pair by DNA polymerase enzymes. Here we examine quantitative steady-state kinetic data from several published studies involving poorly hydrogen-bonding DNA base analogues and adducts, and analyze the results in terms of solvation, hydrogen bonding, and steric effects. We propose a mechanism that can explain the surprising lack of hydrogen-bonding requirement accompanied by significant selectivity in pairing. This hypothesis makes use of steric matching, enforced both by the tightly confined polymerase active site and by the DNA backbone, as a chief factor determining nucleotide selection during DNA synthesis. The results also suggest that hydrogen bonds from bases to water (solvation) may be important in increasing the effective size of DNA bases, which may help prevent misinsertion of small bases opposite each other. © 1998 John Wiley & Sons, Inc. Biopoly 48: 3–17, 1998     

Does polyploidy lead to fewer and shorter microsatellites in Barbus (Teleostei: Cyprinidae)?

Screening of a hybrid Barbus barbus-B. meridionalis genome was performed for CA, GA, TAT, TCT, TAG, TGT, TATT, TACT, ATCT motifs, and simultaneously on another fish species, tilapia S. melanotheron . Sequences of positive clones were obtained for Barbus and revealed that repetitive structure significantly depends on the motif: most TAT and TATT repeats contain small numbers of repeats, and these repeats are highly heterogeneous, whereas other motifs (we mainly obtained CA and GATA repeats) form longer and much more homogeneous arrays. Polymorphism data from five loci in two different species of barbel show that perfectly repetitive loci are much more variable than imperfect loci (TAT and TATT). We compared the frequency of positive clones for different repeat motifs between barbel and tilapia. For dinucleotide repeats (CA and GA), the comparison was extended to additional fish species, trout and sea bass, which were screened in nearly identical conditions for these motifs. The most salient feature of these comparisons reveals that arrays of dinucleotide motifs are significantly under-represented and shorter in Barbus than in other fish species. We propose an explanation that can account for most features of microsatellites characterizing the genome of barbel. A bias toward deletion affecting slipped-strand mispairing events would lead to shortening and loss of microsatellite loci. Such a bias would represent an efficient way of eliminating useless DNA from polyploidized species with an excessive amount of DNA.     

Frequent mitochondrial gene rearrangements at the hymenopteran nad3-nad5 junction

Abstract We characterized the organization of mitochondrial genes from a diverse range of hymenopterans. Of the 21 taxa characterized, 12 had distinct, derived organizations. Some rearrangements were consistent with the duplication–random loss mechanism, while others were not. Local inversions were relatively common, i.e., rearrangements characterized by the movement of genes from one mitochondrial strand to the other, opposite or close to their ancestral position. This type of rearrangement is inconsistent with the duplication/random loss model of mitochondrial gene rearrangement. Instead, they are best explained by the operation of recombination. Taxa with derived organizations were restricted to a single, monophyletic group of wasps, the Apocrita, which comprise about 90% of all hymenopterans.     

Some effects of metal ions on DNA structure and genetic information transfer

The reaction of metal ions with nucleic acids can lead to a variety of dramatic effects on nucleic acid structure, e.g., crosslinking of the polymer strands, degradation to oligomers and monomers, stabilization or destabilization, and the mispairing of bases. These effects have important implications for genetic information transfer. Metal ions are involved in many aspects of this transfer; we are presently concerned with the effect of metal ions on the orientation of the active site of RNA polymerase. Many of the effects of metal ions on nucleic acid structure involve changes in the conformation of the macromolecules. We have found that conditions that have been used to convert B DNA to Z DNA lead to at least two other conformational changes, and phase diagrams delineate the realms of stability of each of the forms. We have carried out a number of studies that demonstrate that the conversion of B to Z DNA is very closely correlated with a substantial decrease in the ability of the DNA to act as a template for RNA synthesis. A portion of this paper has been taken from another paper on “Changes of Biological Significance Induced by Metal Ions in the Structure of Nucleic Acids,” published in Annali dell' lstituto Superiore di Sanita.     

Complete chloroplast genome sequence of Elodea canadensis and comparative analyses with other monocot plastid genomes

Elodea canadensis is an aquatic angiosperm native to North America. It has attracted great attention due to its invasive nature when transported to new areas in its non-native range. We have determined the complete nucleotide sequence of the chloroplast (cp) genome of Elodea. Taxonomically Elodea is a basal monocot, and only few monocot cp genomes representing early lineages of monocots have been sequenced so far. The genome is a circular double-stranded DNA molecule 156,700bp in length, and has a typical structure with large (LSC 86,194bp) and small (SSC 17,810bp) single-copy regions separated by a pair of inverted repeats (IRs 26,348bp each). The Elodea cp genome contains 113 unique genes and 16 duplicated genes in the IR regions. A comparative analysis showed that the gene order and organization of the Elodea cp genome is almost identical to that of Amborella trichopoda, a basal angiosperm. The structure of IRs in Elodea is unique among monocot species with the whole cp genome sequenced. In Elodea and another monocot Lemna minor the borders between IRs and LSC are located upstream of rps19 gene and downstream of trnH-GUG gene, while in most monocots, IR has extended to include both trnH and rps19 genes. A phylogenetic analysis conducted using Bayesian method, based on the DNA sequences of 81 chloroplast genes from 17 monocot taxa provided support for the placement of Elodea together with Lemna as a basal monocot and the next diverging lineage of monocots after Acorales. In comparison with other monocots, the Elodea cp genome has gone through only few rearrangements or gene losses. IR of Elodea has a unique structure among the monocot species studied so far as its structure is similar to that of a basal angiosperm Amborella. This result together with phylogenetic analyses supports the placement of Elodea as a basal monocot to the next diverging lineage of monocots after Acorales. So far, only few cp genomes representing early lineages of monocots have been sequenced and, therefore, this study provides valuable information about the course of evolution in divergence of monocot lineages.     

回文序列诱导突变的机制及人类相关疾病

在原核和真核生物基因组中, 含有回文序列的区域高度可变且稳定性差, 主要原因是回文序列能形成发卡或十字形二级结构, 然后通过滑动错配、单链复性以及非同源末端连接(Non-homologous end joining, NHEJ)等机制导致缺失突变或染色体易位的发生。在人类基因组中, 回文序列较普遍存在于基因表达调控的重要作用元件中, 它诱导的缺失和易位突变还与男性不育、地中海贫血等多种疾病的发生、发展密切相关。文章综合近几年国内外相关文献, 初步阐释回文序列诱导突变的类型和可能机制, 及其与人类疾病的关系, 为进一步探讨回文序列在基因表达调控、基因突变及人类疾病中的作用及功能等相关研究提供参考。     

Structural plasmid instability in Bacillus subtilis: Effect of direct and inverted repeats

Summary Using precise excision as a model system, we have quantified the effect of direct repeats, inverted repeats and the size of the spacer between the repeats in the process of deletion formation in Bacillus subtilis. Both in the presence and absence of inverted repeats, the frequency of precise excision was strongly dependent on the direct repeat length. By increasing the direct repeat length from 9 bp to 18 and 27 bp, the precise excision frequency was raised by 3 and 4 orders of magnitude, respectively. In addition, irrespective of the direct repeat length, the presence of flanking inverted repeats enhanced the excision frequency by 3 orders of magnitude. Varying the inverted repeat length and the spacer size over a wide range did not significantly affect the excision frequencies. These results fit well into a model for deletion formation by slipped mispairing during replication of single-stranded plasmid DNA.     

How slippage-derived sequences are incorporated into rRNA variable-region secondary structure: implications for phylogeny reconstruction

We analyzed the type and frequency of mutational changes in hypervariable rRNA regions, using the highly length-variable region V4 of the small subunit rRNA locus of tiger beetles (Cicindelidae) as an example. Phylogenetic analysis of indels in closely related species showed that (1) most indels are single nucleotides (usually A or T and sometimes G) or di-nucleotides of A and T. These occur at numerous foci, and they exhibit a strong bias for duplication of 5' single and di-nucleotide motifs but not 3' motifs. (2) Insertions/deletions in stem-forming regions affected paired and unpaired bases with about equal frequency but they did not disrupt the secondary structure. (3) Recurring mutations involving short repeats of the same bases caused parallel evolution of similar sequence motifs in the rRNA of different lineages. The observed types of change are consistent with the propostion that slippage is the main mutational mechanism. Slippage-derived sequences tend to be self-complementary, and therefore the stem-loop structure could be self-organizing as a consequence of the underlying mutational mechanism. Thus, the secondary structure in the cicindelid V4 region may be conserved due to the dynamics of the mutational mechanism rather than to functional constraints. These processes may also have a tendency to produce similar primary sequences irrespective of phylogenetic associations. The findings have implications for sequence alignment in phylogenetic analysis and should caution against the use of secondary structure to improve the determination of positional homology in hypervariable regions.     

Crystal structures of A-DNA duplexes

All crystal structures of A-DNA duplexes exhibit a typical crystal packing, with the termini of one molecule abutting the shallow grooves of symmetry related neighbors, while all other forms (B, Z, and RNA) tend to form infinitely stacked helices. The A-DNA arrangement leads to the formation of shallow groove base multiples that have implications for the structure of DNA in compacted states. The characteristic packing leaves big solvent channels, which can be sometimes occupied by B-DNA duplexes. Comparisons of the structures of the same oligomer crystallizing in two different space groups and of different sequences crystallizing in the same space group show that the lattice forces dominate the A-DNA conformation in the crystals, complicating the effort to elucidate the influence of the base sequence on the structures. Nevertheless, in both alternating and nonalternating fragments some sequence effects can still be uncovered. Furthermore, several studies have started to define the minimal sequence changes or chemical modifications that can interconvert the oligomers between different double-helical conformers (A-, B-, and Z-form). Overall, it is seen that the rigid nucleotide principle applies to the oligomeric fragments. Besides the structures of the naked DNAs, their interactions with water, polyamines, and metal ions have attracted considerable attention. There are conserved patterns in the hydration, involving both the grooves and the backbone, which are different from those of B-DNA or Z-DNA. Overall, A-DNA seems to be more economically hydrated than B-DNA, particularly around the sugar-phosphate backbone. Spermine was found to be able to bind exclusively to either of the grooves or to the phosphate groups of the backbone, or exhibit a mixed binding mode. The located metal cations prefer binding to guanine bases and phosphate groups. The only mispairs investigated in A-DNA are the wobble pairs, yielding structural insight into their effects on helix stabilities and hydration. G · T wobble pairs have been determined in various sequence contexts, where they differentially affect the conformations and stableness of the duplexes. The structure of a G · ^m5C base pair, which surprisingly also adopted the wobble conformation, suggests that a similar geometry may transiently exist for G · C pairs. These results from the crystalline state will be compared to the solution state and discussed in relation to their relevance in biology. © 1997 John Wiley & Sons, Inc. Biopoly 44: 45–63, 1997