Repair of Single- and Multiple-Substitution Mismatches during Recombination in Streptococcus Pneumoniae

The use as genetic markers, during transformation of Streptococcus pneumoniae, of 19 sequences differing from wild type, located throughout the amiA locus, enabled us to examine the fate of 24 single- and 11 multiple-mismatches during recombination. Tentative mismatch ranking as a function of decreasing repair efficiency by the Hex mismatch repair system is G/T = A/C = G/G (maximum repair: 90-95%) greater than C/T (mostly 75 to 90% repair) greater than A/A (from 50 to 90% repair) greater than T/T (50-65% repair) greater than A/G (from 0 to 20% repair) greater than C/C. No indication of correction of the latter has been obtained. Over the limited number of samples examined, we observed no influence of the base composition of the surrounding sequence on correction efficiency for both transition mismatches and for G/G and C/C. Variations in the surrounding sequence affect repair of A/G and C/T, and, even more strongly, of A/A and T/T. No simple correlation to the G:C content of the surrounding sequence is apparent from our results, in contrast to the conclusion drawn for the Mut mismatch repair system of Escherichia coli. Examination of the fate of multiple mismatches suggests that C/C may sometimes impede recognition of otherwise corrected mismatches.     

Different base/base mispairs are corrected with different efficiencies and specificities in monkey kidney cells

Mismatches arise during recombination, as errors of DNA replication, and from deamination of 5-methylcytosine to thymine. We determined the efficiency and specificity of mismatch correction in simian cells. Analysis of plaques, obtained after transfection with SV40 DNA molecules harboring a single mispair in a defined orientation within the intron of the large T antigen gene, revealed that all types of base/base mispairs were corrected, albeit with different efficiencies and specificities. Heterogeneous mispairs G/T, A/C, C/T, and A/G, corrected with 96%, 78%, 72%, and 39% efficiencies, respectively, tended to be corrected to G/C. Homogeneous mispairs G/C, C/C, A/A, and T/T were corrected with 92%, 66%, 58%, and 39% efficiencies, respectively, and repair bias was influenced by mismatch flanking sequences.     

Distribution and evolution of sequence characteristics in the E. coli genome

The mean (G + C) composition (51.0%) and standard deviation (+/- 3.8%) of published DNA sequences accounting for 10% of the E. coli genome is in excellent agreement with the principal overall distribution determined by high resolution melting. While differences in base and neighbor characteristics are small and uniform throughout all regions of the genome, it is found that the (G + C) content of sequences varies in segmented fashion within boundaries corresponding to coding (53% G + C) and noncoding (46% G + C) regions; with variances in the latter being six-fold greater than in coding regions. The variance in different regions shows a strong negative dependence on (G + C) content of the region, reflecting the condition that A-T and G-C base pairs are preferred neighbors of A-T and C-G pairs, respectively; with the bias increasing with decreasing (G + C) content. Neighbor analysis indicates the most extreme positive biases occur in AA, TT, GC and CG throughout all regions, but particularly in noncoding regions. Extraordinary numbers of oligomeric strings of (A)n, etc., are the further consequence of this bias. These and other characteristics point to the existence of inherent biases in neighbor frequencies levied during replication or repair, and which reflect, in turn, neighbor influences during mutation. The bias in codon usage noted by Grantham and others is seen here as due, in part, to the adaptation of coding sequences to this microenvironment through selection among synonymous codons so as to preserve inherent neighbor biases.     

Very short patch repair of T:G mismatches in vivo: importance of context and accessory proteins.

In Escherichia coli, T:G mismatches in specific contexts are corrected by a very short patch (VSP) repair system. Previous studies have shown that the product of gene vsr mediates correction of T:G to C:G in the 5'CTAGG/3'GGTCC context and in some related contexts. Amber mutations that arose in CAG sequences in gene cI of bacteriophage lambda were used to determine the effect of flanking bases on the repair of T:G mispairs arising during phage recombination. The experimental findings were combined with published data on mismatch repair of mutations in lambda gene P and E. coli gene lacI. While VSP repair was most efficient in the context 5'CTAGG, there was very significant correction when either the 5'C or the 3' G was replaced by another base. Some mismatch repair of TAG to CAG occurred in all contexts tested. Reduction in VSP repair caused by the lack of MutL or MutS was fully complemented by the addition of vsr+ plasmids when the T:G mispair was in the 5'CTAGG/3'GGTCC context. VSP repair was decreased in bacteria containing mutS+ on a multicopy plasmid. It is suggested that VSP repair maintains sequences such as the repetitive extragenic palindromic (REP) and Chi sequences, which have important roles in E. coli and closely related bacteria.     

Repair of Single Base-Pair Transversion Mismatches of Escherichia Coli in Vitro: Correction of Certain a/G Mismatches Is Independent of Dam Methylation and Host Muthls Gene Functions

Six different base-pair transversion mismatches are repaired with different efficiencies in an in vitro mismatch repair system. In particular, the T/T and C/C mismatches appear to be less efficiently repaired than the A/A and G/G mismatches. Four A/G and four C/T mismatches at different positions are repaired to different extents. One of the A/G mismatches is repaired equally efficiently when DNA heteroduplexes are fully methylated or hemi-methylated at the d(GATC) sequences. This type of mismatch repair appears to be unidirectional with A to C conversion by acting at A/G mispairs to restore the C/G pairs. This methylation-independent correction is not controlled by the mutH, mutL, mutS, uvrE, uvrB, phr, recA, recF, and recJ gene products. The independence of the transversion mismatch repair of these genes and methylation distinguishes this from the known mismatch repair pathways.     

Analysis of Genomic G + C Content, Codon Usage, Initiator Codon Context and Translation Termination Sites In Tetrahymena Thermophila

In recent years, the amount of molecular sequencing data from Tetrahymena thermophila has dramatically increased. We analyzed G + C content, codon usage, initiator codon context and stop codon sites in the extremely A + T rich genome of this ciliate. Average G + C content was 38% for protein coding regions, 21% for 5' non-coding sequences, 19% for 3' non-coding sequences, 15% for introns, 19% for micronuclear limited sequences and 17% for macronuclear retained sequences flanking micronuclear specific regions. The 75 available T. thermophila protein coding sequences favored codons ending in T and, where possible, avoided those with G in the third position. Highly expressed genes were relatively G + C-rich and exhibited an extremely biased pattern of codon usage while developmentally regulated genes were more A + T-rich and showed less codon usage bias. Regions immediately preceding Tetrahymena translation initiator codons were generally A-rich. For the 60 stop codons examined, the frequency of G in the end + 1 site was much higher than expected whereas C never occupied this position.     

Heteroduplex DNA correction in Saccharomyces cerevisiae is mismatch specific and requires functional PMS genes.

In vitro-constructed heteroduplex DNAs with defined mismatches were corrected in Saccharomyces cerevisiae cells with efficiencies that were dependent on the mismatch. Single-nucleotide loops were repaired very efficiently; the base/base mismatches G/T, A/C, G/G, A/G, G/A, A/A, T/T, T/C, and C/T were repaired with a high to intermediate efficiency. The mismatch C/C and a 38-nucleotide loop were corrected with low efficiency. This substrate specificity pattern resembles that found in Escherichia coli and Streptococcus pneumoniae, suggesting an evolutionary relationship of DNA mismatch repair in pro- and eucaryotes. Repair of the listed mismatches was severely impaired in the putative S. cerevisiae DNA mismatch repair mutants pms1 and pms2. Low-efficiency repair also characterized pms3 strains, except that correction of single-nucleotide loops occurred with an efficiency close to that of PMS wild-type strains. A close correlation was found between the repair efficiencies determined in this study and the observed postmeiotic segregation frequencies of alleles with known DNA sequence. This suggests an involvement of DNA mismatch repair in recombination and gene conversion in S. cerevisiae.     

Neighboring-nucleotide effects on the mutation patterns of the rice genome

DNA composition dynamics across genomes of diverse taxonomy is a major subject of genome analyses. DNA composition changes are characteristics of both replication and repair machineries. We investigated 3,611,007 single nucleotide polymorphisms （SNPs） generated by comparing two sequenced rice genomes from distant inbred lines （subspecies）, including those from 242,811 introns and 45,462 protein-coding sequences （CDSs）. Neighboring-nucleotide effects （NNEs） of these SNPs are diverse, depending on structural content-based classifications （genomewide, intronic, and CDS） and sequence context-based categories （A/C, A/G, A/T, C/G, C/T, and G/T substitutions） of the analyzed SNPs. Strong and evident NNEs and nucleotide proportion biases surrounding the analyzed SNPs were observed in 1-3 bp sequences on both sides of an SNP. Strong biases were observed around neighboring nucleotides of protein-coding SNPs, which exhibit a periodicity of three in nucleotide content, constrained by a combined effect of codon-related rules and DNA repair mechanisms. Unlike a previous finding in the human genome, we found negative correlation between GC contents of chromosomes and the magnitude of corresponding bias of nucleotide C at -1 site and G at ＋1 site. These results will further our understanding of the mutation mechanism in rice as well as its evolutionary implications.     

The rate and pattern of nucleotide substitution in Drosophila mitochondrial DNA.

The nucleotide sequences of a segment of mitochondrial DNA (mtDNA) have been determined for nine species or subspecies of the subgenus Drosophila of the genus Drosophila. This segment contains two complete protein-coding genes (i.e., NADH dehydrogenase subunit 1 and cytochrome b) and a transfer RNA gene (tRNA(ser)). The G+C content at third-codon positions for the two protein-coding genes was 1.5 times higher than that in the D. melanogaster species group, which belongs to the subgenus Sophophora. However, there was a substantial difference between the nucleotide frequencies of G and C. The number of nucleotide substitutions per silent site was more than three times higher than that for nuclear DNA, although it was only 60% of that for mammalian mtDNA. Both parametric and nonparametric analyses revealed a strong transition-transversion bias in nucleotide substitution, as was observed in mammalian mtDNA. Moreover, the rate of substitution of A and T for G and C is higher than that for the opposite direction. This bias seems to be responsible for the extremely A+T-rich base composition of Drosophila mtDNA. It is also noted that the rate of transitional change between A and G is higher than that between T and C.     

DNA mismatch correction by Very Short Patch repair may have altered the abundance of oligonucleotides in the E. coli genome.

A base mismatch correction process in E. coli K-12 called Very Short Patch (VSP) repair corrects T:G mismatches to C:G when found in certain sequence contexts. Two of the substrate mismatches (5'-CTWGG/3'-GGW'CC; W = A or T) occur in the context of cytosine methylation in DNA and reduce the mutagenic effects of 5-methylcytosine deamination to thymine. However, VSP repair is also known to repair T:G mismatches that are not expected to arise from 5-methylcytosine deamination (example--CTAG/GGT-C). In these cases, if the original base pair were a T:A, VSP repair would cause a T to C transition. We have carried out Markov chain analysis of an E. coli sequence database to determine if repair at the latter class of sites has altered the abundance of the relevant tetranucleotides. The results are consistent with the prediction that VSP repair would tend to deplete the genome of the 'T' containing sequences (example--CTAG), while enriching it for the corresponding 'C' containing sequences (CCAG). Further, they provide an explanation for the known scarcity of CTAG containing restriction enzyme sites among the genomes of enteric bacteria and identify VSP repair as a force in shaping the sequence composition of bacterial genomes.     

Trends in the 5' vs. 3' flanks of oligonucleotides in eukaryotic and prokaryotic genomes: the asymmetric roles played by cytosine and guanine.

Studies of sequence context preferences of oligonucleotides composed of (G/C)n and (A/T)m blocks (n + m = 3,4,5) unravel strong patterns. Comparisons of the 5' and 3' nearest neighbor doublets flanking these oligomers reveal the preference of (G/C)2 to be positioned immediately next to the (A/T)m block, enclosing it by (G/C) nucleotides rather than extending the (G/C)n block. That is, for a (G/C)n(A/T)m oligomer and a (G/C)2 doublet, (G/C)n(A/T)m(G/C)2 greater than (G/C)n + 2 (A/T)m. Similarly for an (A/T)m(G/C)n oligomer, (G/C)2(A/T)m(G/C)n greater than (A/T)m(G/C)n + 2. In an analogous manner, (A/T)2 flanking doublets prefer enclosing the (G/C)n blocks, although these patterns are weaker. Here we show a strong, direct relationship between the magnitude of the trends and the presence of Cs in the (G/C)n block in the (G/C)n(A/T)m oligomer, and the presence of Gs in the complementary (A/T)m(G/C)n oligomers. The trends are stronger in eukaryotic than in prokaryotic sequences. They are stronger for longer (G/C)n and shorter (A/T)m blocks. We suggest that the preference for (A/T)m to be enclosed by (G/C) rather than be flanked by them on only one side is related to DNA structure and DNA-protein interaction. Sequences of the (G/C)(A/T)(G/C) type may have more homogeneous minor groove geometry. In particular, the strong G vs. C asymmetry in the trends may be related to pyrimidine-purine junctions, possibly to CG sequences.     

Stability and mismatch discrimination of locked nucleic acid-DNA duplexes

Locked nucleic acids (LNA; symbols of bases, +A, +C, +G, and +T) are introduced into chemically synthesized oligonucleotides to increase duplex stability and specificity. To understand these effects, we have determined thermodynamic parameters of consecutive LNA nucleotides. We present guidelines for the design of LNA oligonucleotides and introduce free online software that predicts the stability of any LNA duplex oligomer. Thermodynamic analysis shows that the single strand-duplex transition is characterized by a favorable enthalpic change and by an unfavorable loss of entropy. A single LNA modification confines the local conformation of nucleotides, causing a smaller, less unfavorable entropic loss when the single strand is restricted to the rigid duplex structure. Additional LNAs adjacent to the initial modification appear to enhance stacking and H-bonding interactions because they increase the enthalpic contributions to duplex stabilization. New nearest-neighbor parameters correctly forecast the positive and negative effects of LNAs on mismatch discrimination. Specificity is enhanced in a majority of sequences and is dependent on mismatch type and adjacent base pairs; the largest discriminatory boost occurs for the central +C·C mismatch within the +T+C+C sequence and the +A·G mismatch within the +T+A+G sequence. LNAs do not affect specificity in some sequences and even impair it for many +G·T and +C·A mismatches. The level of mismatch discrimination decreases the most for the central +G·T mismatch within the +G+G+C sequence and the +C·A mismatch within the +G+C+G sequence. We hypothesize that these discrimination changes are not unique features of LNAs but originate from the shift of the duplex conformation from B-form to A-form.     

DNA-substrate sequence specificity of human G:T mismatch repair activity.

G:T mispairs in DNA originate spontaneously via deamination of 5-methylcytosine. Such mispairs are restored to normal G:C pairs by both E. coli K strains and human cells. In this study we have analyzed the repair by human cell extracts of G:T mismatches in various DNA contexts. We performed two sets of experiments. In the first, repair was sequence specific in that G:T mispairs at CpG sites at four different CpG sites were repaired, but a G:T mismatch at a GpG site was not. Cytosine hemimethylation did not block repair of a substrate containing a CpG/GpT mismatch. In the second set of experiments, substrates with a G:T mismatch at a fixed position were constructed with an A, T, G, or C 5' to the mismatched G, and alterations in the complementary strand to allow otherwise perfect Watson-Crick pairing. All were incised just 5' to the mismatched T and competed for repair incision with a G:T substrate in which a C was 5' to the mismatched G. Thus human G:T mismatch activity shows sequence specificity, incising G:T mismatched pairs at some DNA sites, but not at others. At an incisable site, however, incision is little influenced by the base 5' to the mismatched G.     

Gene mutations of 23S rRNA associated with clarithromycin resistance in Helicobacter pylori strains isolated from Korean patients

Although resistance of Helicobacter pylori to clarithromycin is a major cause of failure of eradication therapies, little information is available regarding gene mutations of clarithromycin-resistant primary and secondary H. pylori isolates in Korea. In the present study, we examined gene mutations of H. pylori 23S rRNA responsible for resistance to clarithromycin. DNA sequences of the 23S rRNA gene in 21 primary clarithromycin-resistant and 64 secondary clarithromycin-resistant strains were determined by PCR amplification and nucleotide sequence analyses. Two mutations of the 23S rRNA gene, A2143G and T2182C, were observed in primary clarithromycin-resistant isolates. In secondary isolates, dual mutation of A2143G+T2182C was frequently observed. In addition, A2143G+T2182C+ T2190C, A2143G+T2182C+C2195T, and A2143G+T2182C +A2223G were observed in secondary isolates. Furthermore, macrolide binding was tested on purified ribosomes isolated from T2182C or A2143C mutant strains with [14C]erythromycin. Erythromycin binding increased in a dose-dependent manner for the susceptible strain but not for the mutant strains. These results indicate that secondary isolates show a greater variety of 23S rRNA gene mutation types than primary isolates, and triple mutations of secondary isolates are associated with A2143G+T2182C in H. pylori isolated from Korean patients.     

Base mismatch-specific endonuclease activity in extracts from Saccharomyces cerevisiae.

An endonuclease activity (called MS-nicking) for all possible base mismatches has been detected in the extracts of yeast, Saccharomyces cerevisiae. DNAs with twelve possible base mismatches at one defined position are cleaved at different efficiencies. DNA fragments with A/G, G/A, T/G, G/T, G/G, or A/A mismatches are nicked with greater efficiencies than C/T, T/C, C/A, and C/C. DNA with an A/C or T/T mismatch is nicked with an intermediate efficiency. The MS-nicking is only on one particular DNA strand, and this strand disparity is not controlled by methylation, strand break, or nature of the mismatch. The nicks have been mapped at 2-3 places at second, third, and fourth phosphodiester bonds 5' to the mispaired base; from the time course study, the fourth phosphodiester bond probably is the primary incision site. This activity may be involved in mismatch repair during genetic recombination.     

云南保山猪线粒体DNA D-loop区序列初步分析 Primary Analysis of Mitochondrial DNA D-loop Region Sequence in Baoshan Pig

摘要：为了解云南保山猪(Baoshan pig)的遗传多样性及其遗传背景,我们测定了19个个体线粒体DNA D-loop高变区I 15 363～15 801片段序列438 bp。检测到10种单倍型,包括8个多态位点,其中5次T/C转换、1次G/A转换、1次G/C颠换和1次A/T颠换,其A、T、G、C碱基的平均含量分别为35.4%、26.9%、13.2%和24.5%,A+T含量(62.3%)明显高于G+C含量(37.7%)。对于保山猪的保种及其持续利用有着重要的理论指导意义。 Abstract:To investigate the genetic diversity and genetic data of Baoshan pig in Yunnan province,the mitochondrial DNA D-loop hypervariable segment I sequences 15 363～15 801 (438 bp) in 19 individuals of Baoshan pig were sequenced.Ten mitochondrial haplotypes were identified in the samples,with 8 sites showing polymorphism,which were 5 T/C and 1 G/A transitions,1 G/C and 1 A/T transversions.The contents of A,T,G and C were 35.4%,269%,13.2% and 24.5%,respectively.The content of A+T (62.3%) was significantly higher than that of G+C (37.3%).It will be of importance to conservation and sustainable utilization in Baoshan pig.     

Mutator activity and specificity of Escherichia coli dnaQ49 allele--effect of umuDC products

The high fidelity of DNA replication in Escherichia coli is ensured by the alpha (DnaE) and epsilon (DnaQ) subunits of DNA polymerase providing insertion fidelity, 3'-->5' exonuclease proofreading activity, and by the dam-directed mismatch repair system. dnaQ49 is a recessive allele that confers a temperature-sensitive proofreading phenotype resulting in a high rate of spontaneous mutations and chronic induction of the SOS response. The aim of this study was to analyse the mutational specificity of dnaQ49 in umuDC and DeltaumuDC backgrounds at 28 and 37 degrees C in a system developed by J.H. Miller. We confirmed that the mutator activity of dnaQ49 was negligible at 28 degrees C and fully expressed at 37 degrees C. Of the six possible base pair substitutions, only GC-->AT transitions and GC-->TA and AT-->TA transversions were appreciably increased. However, the most numerous mutations were frameshifts, -1G deletions and +1A insertions. All mutations which increased in response to dnaQ49 damage were to a various extent umuDC-dependent, especially -1G deletions. This type of mutations decreased in CC108dnaQ49DeltaumuDC to 10% of the value found in CC108dnaQ49umuDC+ and increased in the presence of plasmids producing UmuD'C or UmuDC proteins. In the recovery of dnaQ49 mutator activity the plasmid harbouring umuD'C genes was more effective than the one harbouring umuDC. Analysis of mutational specificity of pol III with defective epsilon subunit indicates that continuation of DNA replication is allowed past G:T, C:T, T:T (or C:A, G:A, A:A) mismatches but does not allow for acceptance of T:C, C:C, A:C (or A:G, G:G, T:G) (the underlined base is in the template strand).     

基于mtDNA COI基因的系统分析表明栗属植物上隐藏瘿蜂种的存在

本研究利用mtDNACOI基因的部分序列对11种群不同寄主的栗瘿蜂Dryocosm us kuriphilus Yasumatsu遗传多态性进行了分析。在获得的660bp的序列中,变异位点(V)80个,包括简约信息位点(Pi)15个和自裔位点(Si)65个。T、A、C、G平均含量分别为30.6%、43.3%、13.7%和12.4%,A+T平均值为73.9%,远高于G+C的26.1%,表现出明显的A+T偏向性。为害锥栗的六步溪、大围山种群与茅栗、板栗的不同地理种群,以及为害欧洲栗的意大利Piedmont种群之间,基因序列完全一致或者遗传距离在0.3%以内,无论是NJ系统树还是UPGMA系统树均聚合在同一分支内,亲缘关系较近。而为害锥栗的采自福建省建瓯市水源镇的水源种群、福建省周宁县纯池镇的纯池种群,与其它各栗瘿蜂种群的基因序列差异较大,遗传距离均大于2%,在NJ和UPGMA系统树中水源种群和纯池种群均聚合为一单独的分支,与其它种群形成的分支互为姊妹分支。据此我们认为水源种群和纯池种群很有可能属于不同的物种,有进一步研究的必要。     

The longest (A+T) and (G+C) blocks in the human and other genomes

We analysed complete or almost complete nucleotide sequences of the human, chimp, mouse, rat, chicken, dog, and other genomes to find that they contain extremely long (A+T) a (G+C) blocks that do not occur at all in the corresponding randomized sequences. The longest is an (A+T) block containing 1040 consecutive AT pairs that occurs in the 16th human chromosome. The longest human (G+C) block has 261 bp in length. About a half of the longest blocks occur in introns. The (A+T) blocks are discrete units whereas the (G+C) blocks are diffuse. They are imbedded in the genome through connectors longer than 1 kilobase where the (G+C) content gradually decreases to the value of 50%. Remarkably, the (A+T) as well as (G+C) blocks are substantially shorter in the chimp genome. Chicken is characteristic by very long (G+C) blocks that are even longer than in the human genome. Though much shorter, long (G+C) and especially (A+T) blocks occur in lower organisms as well, which means that AT and GC pair clustering is an ancient property that has evolved into large scales in higher eukaryote genomes and the human genome in particular. Very long (A+T) and (G+C) blocks confer specific biophysical properties on DNA that are likely to influence genome folding in cell nuclei and its functional properties.