Higher mutation rate helps to rescue genes from the elimination by selection

Directional mutation pressure associated with replication processes is the main cause of the asymmetry between the leading and lagging DNA strands in bacterial genomes. On the other hand, the asymmetry between sense and antisense strands of protein coding sequences is a result of both mutation and selection pressures. Thus, there are two different ways of superposition of the sense strand, on the leading or lagging strand. Besides many other implications of these two possible situations, one seems to be very important - because of the asymmetric replication-associated mutation pressure, the mutation rate of genes depends on their location. Using Monte Carlo methods, we have simulated, under experimentally determined directional mutation pressure, the divergence rate and the elimination rate of genes depending on their location in respect to the leading/lagging DNA strands in the asymmetric prokaryotic genome. We have found that the best survival strategy for the majority of genes is to sometimes switch between DNA strands. Paradoxically, this strategy results in higher substitution rates but remains in agreement with observations in bacterial genomes that such inversions are very frequent and divergence rate between homologs lying on different DNA strands is very high.     

Is there a role for replication fork asymmetry in the distribution of genes in bacterial genomes?

Replication generates bacterial chromosomes with strands that differ in the number of genes and base composition. It has been suggested that in bacteria such as Bacillus subtilis, PolC is responsible for the synthesis of the leading strand and DnaE for the lagging strand, whereas in many other bacteria DnaE is responsible for the synthesis of both strands. Here, I show that the possession of PolC correlates with leading strands that contain an average of 78% of genes compared with 58% for genomes that do not contain PolC. This suggests that asymmetrical replication forks could have a major role in defining and constraining the structure of the bacterial chromosome. The presence of PolC is not correlated with compositional strand bias, suggesting that the two biases result from different types of structural asymmetry.     

Codon usage and gene expression pattern of Stenotrophomonas maltophilia R551-3 for pathogenic mode of living

Stenotrophomonas maltophilia strain R551-3 is a multiple-antibiotic-resistant opportunistic human pathogen involved in nosocomial infections. It has a widely distributed GC-rich (>66%) genome. Analysis of differential expression of the genes of this genome reveals that majority of genes belonging to highly expressed category are mostly present on lagging strand without showing any strand specific codon usage bias. Relatively small number of lowly expressed genes is equally distributed on both leading and lagging strands with a difference in codon usage pattern between them. Among several multi drug resistance genes of S. maltophilia involving lowly expressed category some are predicted as horizontally transferred. It can be inferred that horizontally transferred genes may have been imported into this genome for their pathogenic mode of living. Our study may help to modify the expression level of the target genes of this human pathogen in order to control its infection.     

The Differential Killing of Genes by Inversions in Prokaryotic Genomes

We have elaborated a method which has allowed us to estimate the direction of translocation of orthologs which have changed, during the phylogeny, their positions on chromosome in respect to the leading or lagging role of DNA strands. We have shown that the relative number of translocations which have switched positions of genes from the leading to the lagging DNA strand is lower than the number of translocations which have transferred genes from the lagging strand to the leading strand of prokaryotic genomes. This paradox could be explained by assuming that the stronger mutation pressure and selection after inversion preferentially eliminate genes transferred from the leading to the lagging DNA strand. Received: 12 December 2000 / Accepted: 20 April 2001     

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.     

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.     

Proteome composition and codon usage in spirochaetes: species-specific and DNA strand-specific mutational biases

The genomes of the spirochaetes Borrelia burgdorferi and Treponema pallidum show strong strand-specific skews in nucleotide composition, with the leading strand in replication being richer in G and T than the lagging strand in both species. This mutation bias results in codon usage and amino acid composition patterns that are significantly different between genes encoded on the two strands, in both species. There are also substantial differences between the species, with T.pallidum having a much higher G+C content than B. burgdorferi. These changes in amino acid and codon compositions represent neutral sequence change that has been caused by strong strand- and species-specific mutation pressures. Genes that have been relocated between the leading and lagging strands since B. burgdorferi and T.pallidum diverged from a common ancestor now show codon and amino acid compositions typical of their current locations. There is no evidence that translational selection operates on codon usage in highly expressed genes in these species, and the primary influence on codon usage is whether a gene is transcribed in the same direction as replication, or opposite to it. The dnaA gene in both species has codon usage patterns distinctive of a lagging strand gene, indicating that the origin of replication lies downstream of this gene, possibly within dnaN. Our findings strongly suggest that gene-finding algorithms that ignore variability within the genome may be flawed.     

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     

Coordinated leading and lagging strand DNA synthesis by using the herpes simplex virus 1 replication complex and minicircle DNA templates

The origin-specific replication of the herpes simplex virus 1 genome requires seven proteins: the helicase-primase (UL5-UL8-UL52), the DNA polymerase (UL30-UL42), the single-strand DNA binding protein (ICP8), and the origin-binding protein (UL9). We reconstituted these proteins, excluding UL9, on synthetic minicircular DNA templates and monitored leading and lagging strand DNA synthesis using the strand-specific incorporation of dTMP and dAMP. Critical features of the assays that led to efficient leading and lagging stand synthesis included high helicase-primase concentrations and a lagging strand template whose sequence resembled that of the viral DNA. Depending on the nature of the minicircle template, the replication complex synthesized leading and lagging strand products at molar ratios varying between 1:1 and 3:1. Lagging strand products (～0.2 to 0.6 kb) were significantly shorter than leading strand products (～2 to 10 kb), and conditions that stimulated primer synthesis led to shorter lagging strand products. ICP8 was not essential; however, its presence stimulated DNA synthesis and increased the length of both leading and lagging strand products. Curiously, human DNA polymerase α (p70-p180 or p49-p58-p70-p180), which improves the utilization of RNA primers synthesized by herpesvirus primase on linear DNA templates, had no effect on the replication of the minicircles. The lack of stimulation by polymerase α suggests the existence of a macromolecular assembly that enhances the utilization of RNA primers and may functionally couple leading and lagging strand synthesis. Evidence for functional coupling is further provided by our observations that (i) leading and lagging strand synthesis produce equal amounts of DNA, (ii) leading strand synthesis proceeds faster under conditions that disable primer synthesis on the lagging strand, and (iii) conditions that accelerate helicase-catalyzed DNA unwinding stimulate decoupled leading strand synthesis but not coordinated leading and lagging strand synthesis.     

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.     

Selection for Chromosome Architecture in Bacteria

Bacterial chromosomes are immense polymers whose faithful replication and segregation are crucial to cell survival. The ability of proteins such as FtsK to move unidirectionally toward the replication terminus, and direct DNA translocation into the appropriate daughter cell during cell division, requires that bacterial genomes maintain an architecture for the orderly replication and segregation of chromosomes. We suggest that proteins that locate the replication terminus exploit strand-biased sequences that are overrepresented on one DNA strand, and that selection increases with decreased distance to the replication terminus. We report a generalized method for detecting these architecture imparting sequences (AIMS) and have identified AIMS in nearly all bacterial genomes. Their increased abundance on leading strands and decreased abundance on lagging strands toward replication termini are not the result of changes in mutational bias; rather, they reflect a gradient of long-term positive selection for AIMS. The maintenance of the pattern of AIMS across the genomes of related bacteria independent of their positions within individual genes suggests a well-conserved role in genome biology. The stable gradient of AIMS abundance from replication origin to terminus suggests that the replicore acts as a target of selection, where selection for chromosome architecture results in the maintenance of gene order and in the lack of high-frequency DNA inversion within replicores. [Reviewing Editor: Dr. Martin Kreitman]     

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.     

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.     

Overlapping sequences with high homology to functional proteins coexist on complementary strands of DNA in the rumen bacterium Prevotella albensis.

The potential for two complementary fragments of DNA from a clone from the ruminal bacterium Prevotella albensis to encode sequences with homology to at least part of functional proteins is described. One strand contains a sequence with high homology to dnaK, a member of the hsp70 family, and the other strand contains a sequence with some homology to glutamate dehydrogenase genes. Overlapping of these two genes on opposite strands has been reported in eukaryotic species, and is now reported for the first time in a bacterial species. Further investigation of previously described dnaK genes demonstrates that it is more widespread than might be anticipated, with all thirty other dnaK genes investigated also retaining long sequences encoding at least part of a sequence with high homology to a glutamate dehydrogenase gene.     

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.