Universal replication biases in bacteria

Analysis of 15 complete bacterial chromosomes revealed important biases in gene organization. Strong compositional asymmetries between the genes lying on the leading versus lagging strands were observed at the level of nucleotides, codons and, surprisingly, amino acids. For some species, the bias is so high that the sole knowledge of a protein sequence allows one to predict with almost no errors whether the gene is transcribed from one strand or the other. Furthermore, we show that these biases are not species specific but appear to be universal. These findings may have important consequences in our understanding of fundamental biological processes in bacteria, such as replication fidelity, codon usage in genes and even amino acid usage in proteins.     

Fidelity of replication of the leading and the lagging DNA strands opposite N-methyl-N-nitrosourea-induced DNA damage in human cells.

Semi-conservative replication of double-stranded DNA in eukaryotic cells is an asymmetric process involving leading and lagging strand synthesis and different DNA polymerases. We report a study to analyze the effect of these asymmetries when the replication machinery encounters alkylation-induced DNA adducts. The model system is an EBV-derived shuttle vector which replicates in synchrony with the host human cells and carries as marker gene the bacterial gpt gene. A preferential distribution of N-methyl-N-nitrosourea (MNU)-induced mutations in the non transcribed DNA strand of the shuttle vector pF1-EBV was previously reported. The hypermutated strand was the leading strand. To test whether the different fidelity of DNA polymerases synthesizing the leading and the lagging strands might contribute to MNU-induced mutation distribution the mutagenesis study was repeated on the shuttle vector pTF-EBV which contains the gpt gene in the inverted orientation. We show that the base substitution error rates on an alkylated substrate are similar for the replication of the leading and lagging strands. Moreover, we present evidence that the fidelity of replication opposite O6-methylguanine adducts of both the leading and lagging strands is not affected by the 3' flanking base. The preferential targeting of mutations after replication of alkylated DNA is mainly driven by the base at the 5' side of the G residues.     

The Effect of Multiple Evolutionary Selections on Synonymous Codon Usage of Genes in the Mycoplasma bovis Genome

Mycoplasma bovis is a major pathogen causing arthritis, respiratory disease and mastitis in cattle. A better understanding of its genetic features and evolution might represent evidences of surviving host environments. In this study, multiple factors influencing synonymous codon usage patterns in M. bovis (three strains’ genomes) were analyzed. The overall nucleotide content of genes in the M. bovis genome is AT-rich. Although the G and C contents at the third codon position of genes in the leading strand differ from those in the lagging strand (p<0.05), the 59 synonymous codon usage patterns of genes in the leading strand are highly similar to those in the lagging strand. The over-represented codons and the under-represented codons were identified. A comparison of the synonymous codon usage pattern of M. bovis and cattle (susceptible host) indicated the independent formation of synonymous codon usage of M. bovis. Principal component analysis revealed that (i) strand-specific mutational bias fails to affect the synonymous codon usage pattern in the leading and lagging strands, (ii) mutation pressure from nucleotide content plays a role in shaping the overall codon usage, and (iii) the major trend of synonymous codon usage has a significant correlation with the gene expression level that is estimated by the codon adaptation index. The plot of the effective number of codons against the G+C content at the third codon position also reveals that mutation pressure undoubtedly contributes to the synonymous codon usage pattern of M. bovis. Additionally, the formation of the overall codon usage is determined by certain evolutionary selections for gene function classification (30S protein, 50S protein, transposase, membrane protein, and lipoprotein) and translation elongation region of genes in M. bovis. The information could be helpful in further investigations of evolutionary mechanisms of the Mycoplasma family and heterologous expression of its functionally important proteins.     

Misincorporation rate and type on the leading and lagging strands of UV-damaged DNA.

We have examined the fidelity of replication of the leading and lagging strands of UV-irradiated DNA by using an EBV-derived shuttle vector system which contains as marker gene for mutation analysis the bacterial gpt gene in both orientations relative to the EBV oriP. Human cells stably transformed with this vector were UV irradiated and gpt mutation rate and type were analysed. An increased mutagenicity associated with UV irradiation was observed, but the average error frequency was unaffected by the direction of replication of the target gene. Some variability by position and sequence context of leading and lagging strand errors was detected, suggesting that the different architecture of the replication complex for the two strands might, to some extent, affect mutation spectra. The comparable fidelity of translesion replication on the leading and lagging strands is in agreement with the current model for eukaryotic replication that postulates the simultaneous synthesis of both strands by a DNA polymerase with a proof-reading exonuclease.     

Asymmetric directional mutation pressures in bacteria

Background

When there are no strand-specific biases in mutation and selection rates (that is, in the substitution rates) between the two strands of DNA, the average nucleotide composition is theoretically expected to be A = T and G = C within each strand. Deviations from these equalities are therefore evidence for an asymmetry in selection and/or mutation between the two strands. By focusing on weakly selected regions that could be oriented with respect to replication in 43 out of 51 completely sequenced bacterial chromosomes, we have been able to detect asymmetric directional mutation pressures.

Results

Most of the 43 chromosomes were found to be relatively enriched in G over C and T over A, and slightly depleted in G+C, in their weakly selected positions (intergenic regions and third codon positions) in the leading strand compared with the lagging strand. Deviations from A = T and G = C were highly correlated between third codon positions and intergenic regions, with a lower degree of deviation in intergenic regions, and were not correlated with overall genomic G+C content.

Conclusions

During the course of bacterial chromosome evolution, the effects of asymmetric directional mutation pressures are commonly observed in weakly selected positions. The degree of deviation from equality is highly variable among species, and within species is higher in third codon positions than in intergenic regions. The orientation of these effects is almost universal and is compatible in most cases with the hypothesis of an excess of cytosine deamination in the single-stranded state during DNA replication. However, the variation in G+C content between species is influenced by factors other than asymmetric mutation pressure.

     

Differential DNA secondary structure-mediated deletion mutation in the leading and lagging strands.

The frequencies of deletion of short sequences (mutation inserts) inserted into the chloramphenicol acetyl-transferase (CAT) gene were measured for pBR325 and pBR523, in which the orientation of the CAT gene was reversed, in Escherichia coli. Reversal of the CAT gene changes the relationship between the transcribed strand and the leading and lagging strands of the DNA replication fork in pBR325-based plasmids. Deletion of these mutation inserts may be mediated by slipped misalignment during DNA replication. Symmetrical sequences, in which the same potential DNA structural misalignment can form in both the leading and lagging strands, exhibited an approximately twofold difference in the deletion frequencies upon reversal of the CAT gene. Sequences that contained an inverted repeat that was asymmetric with respect to flanking direct repeats were designed. With asymmetric mutation inserts, different misaligned structural intermediates could form in the leading and lagging strands, depending on the orientation of the insert and/or of the CAT gene. When slippage could be stabilized by a hairpin in the lagging strand, thereby forming a three-way junction, deletion occurred by up to 50-fold more frequently than when this structure formed in the leading strand. These results support the model that slipped misalignment involving DNA secondary structure occurs preferentially in the lagging strand during DNA replication.     

76种细菌DNA双链碱基使用频率的比较及其意义

应用生物信息学方法,对已完成测序的76种细菌基因组进行比较,分析细菌基因组中编码区及密码子上碱基使用频率情况,结果显示：1.先导链与滞后链上在编码区的碱基使用频率无明显差异且显著正相关;2.先地链与滞后链在第一,第二,第三密码子碱基使用频率基本一致且显著正相关,结果表明,选择压力及自然突变对DNA双链总体碱基分布的影响相等。     

GC skew in protein-coding genes between the leading and lagging strands in bacterial genomes: new substitution models incorporating strand bias

The DNA strands in most prokaryotic genomes experience strand-biased spontaneous mutation, especially C→T mutations produced by deamination that occur preferentially in the leading strand. This has often been invoked to account for the asymmetry in nucleotide composition, typically measured by GC skew, between the leading and the lagging strand. Casting such strand asymmetry in the framework of a nucleotide substitution model is important for understanding genomic evolution and phylogenetic reconstruction. We present a substitution model showing that the increased C→T mutation will lead to positive GC skew in one strand but negative GC skew in the other, with greater C→T mutation pressure associated with greater differences in GC skew between the leading and the lagging strand. However, the model based on mutation bias alone does not predict any positive correlation in GC skew between the leading and lagging strands. We computed GC skew for coding sequences collinear with the leading and lagging strands across 339 prokaryotic genomes and found a strong and positive correlation in GC skew between the two strands. We show that the observed positive correlation can be satisfactorily explained by an improved substitution model with one additional parameter incorporating a general trend of C avoidance.     

Replication Orientation Affects the Rate and Direction of Bacterial Gene Evolution

In many bacterial genomes, the leading and lagging strands have different skews in base composition; for example, an excess of guanosine compared to cytosine on the leading strand. We find that Chlamydia genes that have switched their orientation relative to the direction of replication, for example by inversion, acquire the skew of their new ``host' strand. In contrast to most evolutionary processes, which have unpredictable effects on the sequence of a gene, replication-related skews reflect a directional evolutionary force that causes predictable changes in the base composition of switched genes, resulting in increased DNA and amino acid sequence divergence. Received: 27 April 2000 / Accepted: 1 August 2000     

Increased and Imbalanced dNTP Pools Symmetrically Promote Both Leading and Lagging Strand Replication Infidelity

The fidelity of DNA replication requires an appropriate balance of dNTPs, yet the nascent leading and lagging strands of the nuclear genome are primarily synthesized by replicases that differ in subunit composition, protein partnerships and biochemical properties, including fidelity. These facts pose the question of whether imbalanced dNTP pools differentially influence leading and lagging strand replication fidelity. Here we test this possibility by examining strand-specific replication infidelity driven by a mutation in yeast ribonucleotide reductase, rnr1-Y285A, that leads to elevated dTTP and dCTP concentrations. The results for the CAN1 mutational reporter gene present in opposite orientations in the genome reveal that the rates, and surprisingly even the sequence contexts, of replication errors are remarkably similar for leading and lagging strand synthesis. Moreover, while many mismatches driven by the dNTP pool imbalance are efficiently corrected by mismatch repair, others are repaired less efficiently, especially those in sequence contexts suggesting reduced proofreading due to increased mismatch extension driven by the high dTTP and dCTP concentrations. Thus the two DNA strands of the nuclear genome are at similar risk of mutations resulting from this dNTP pool imbalance, and this risk is not completely suppressed even when both major replication error correction mechanisms are genetically intact.     

The fidelity of the human leading and lagging strand DNA replication apparatus with 8-oxodeoxyguanosine triphosphate.

A product of oxidative metabolism, 8-oxodeoxyguanosine triphosphate (8-O-dGTP), readily pairs with adenine during DNA replication, ultimately causing A.T-->C.G transversions. This study utilized 8-O-dGTP as a probe to examine the fidelity of the leading and lagging strand replication apparatus in extracts of HeLa cells. Simian virus (SV) 40 T antigen-dependent DNA replication reactions were performed with two M13mp2 vectors with the SV40 origin located on opposite sides of the lacZ alpha sequence used to score replication errors. The presence of 8-O-dGTP at equimolar concentration with each of the 4 normal dNTPs resulted in a > 46-fold increase in error rate for A.T-->C.G transversion over that observed in the absence of 8-O-dGTP. A similar average error rate was observed on the (+) and (-) strands in both vectors, suggesting that the fidelity of replication by leading and lagging strand replication proteins is similar for the dA.8-O-dGMP mispair. Replication fidelity in the presence of 8-O-dGTP was reduced on both strands when an inhibitor of exonucleolytic proofreading (dGMP) was added to the reaction. These data suggest that the majority of dA.8-O-dGMP mispairs are proofread by both leading and lagging strand replication proteins.     

Duplications between direct repeats stabilized by DNA secondary structure occur preferentially in the leading strand during DNA replication

To ascertain a leading or lagging strand preference for duplication mutations, several short DNA sequences, i.e. mutation inserts, were designed that should demonstrate an asymmetric propensity for duplication mutations in the two complementary DNA strands during replication. The design of the mutation insert involved a 7-bp quasi inverted repeat that forms a remarkably stable hairpin in one DNA strand, but not the other. The inverted repeat is asymmetrically placed between flanking direct repeats. This sequence was cloned into a modified chloramphenicol acetyltransferase (CAT) gene containing a −1 frameshift mutation. Duplication of the mutation insert restores the reading frame of the CAT gene resulting in a chloramphenicol resistant phenotype. The mutation insert showed greater than a 200-fold preference for duplication mutations during leading strand, compared with lagging strand, replication. This result suggests that misalignment stabilized by DNA secondary structure, leading to duplication between direct repeats, occurred preferentially during leading strand synthesis.