Abstract:Replication-transcription conflicts promote mutagenesis and give rise to evolutionary signatures, with fundamental importance to genome stability ranging from bacteria to metastatic cancer cells. This review focuses on the interplay between replication-transcription conflicts and the evolution of gene directionality. In most bacteria, the majority of genes are encoded on the leading strand of replication such that their transcription is co-directional with the direction of DNA replication fork movement. This g… Show more
“…This is the same bias found in the experiments with lacI (58). Thus, as previously noted (5), when a gene is inverted the DNA context met by the replication machinery for each strand is different, and this fact could result in the mutational biases reported. These biases would be particularly strong when only one or a few specific mutations are scored, as in the reversion assays referenced above.…”
Section: Discussionmentioning
confidence: 67%
“…Several reports have emphasized that the mutations induced by replication-transcription conflicts have signatures [reviewed in (5)]. In both E. coli (16) and B. subtilis (15), inverting a gene revealed orientation-specific hotspots for both BPSs and large duplications and deletions.…”
Encounters between DNA replication and transcription can cause genomic disruption, particularly when the two meet head-on. Whether these conflicts produce point mutations is debated. This paper presents detailed analyses of a large collection of mutations generated during mutation accumulation experiments with mismatch-repair (MMR) defective Escherichia coli. With MMR absent, mutations are primarily due to DNA replication errors. Overall, there were no differences in the frequencies of base-pair substitutions or small indels (insertion and deletions ≤ 4 bp) in the coding sequences or promoters of genes oriented codirectionally versus head-on to replication. Among a subset of highly expressed genes there was a 2- to 3-fold bias for indels in genes oriented head-on to replication, but this difference was almost entirely due to the asymmetrical genomic locations of tRNA genes containing mononucleotide runs, which are hotspots for indels.No additional orientation bias in mutation frequencies occurred when MMR-strains were also defective for transcription-coupled repair (TCR). However, in contrast to other reports, loss of TCR slightly increased the overall mutation rate, meaning that TCR is antimutagenic. There was no orientation bias in mutation frequencies among the stress-response genes that are regulated by RpoS or induced by DNA damage. Thus, biases in the locations of mutational targets can account for most, if not all, apparent biases in mutation frequencies between genes oriented head-on versus co-directional to replication. In addition, the data revealed a strong correlation of the frequency of base-pair substitutions with gene length, but no correlation with gene expression levels.
“…This is the same bias found in the experiments with lacI (58). Thus, as previously noted (5), when a gene is inverted the DNA context met by the replication machinery for each strand is different, and this fact could result in the mutational biases reported. These biases would be particularly strong when only one or a few specific mutations are scored, as in the reversion assays referenced above.…”
Section: Discussionmentioning
confidence: 67%
“…Several reports have emphasized that the mutations induced by replication-transcription conflicts have signatures [reviewed in (5)]. In both E. coli (16) and B. subtilis (15), inverting a gene revealed orientation-specific hotspots for both BPSs and large duplications and deletions.…”
Encounters between DNA replication and transcription can cause genomic disruption, particularly when the two meet head-on. Whether these conflicts produce point mutations is debated. This paper presents detailed analyses of a large collection of mutations generated during mutation accumulation experiments with mismatch-repair (MMR) defective Escherichia coli. With MMR absent, mutations are primarily due to DNA replication errors. Overall, there were no differences in the frequencies of base-pair substitutions or small indels (insertion and deletions ≤ 4 bp) in the coding sequences or promoters of genes oriented codirectionally versus head-on to replication. Among a subset of highly expressed genes there was a 2- to 3-fold bias for indels in genes oriented head-on to replication, but this difference was almost entirely due to the asymmetrical genomic locations of tRNA genes containing mononucleotide runs, which are hotspots for indels.No additional orientation bias in mutation frequencies occurred when MMR-strains were also defective for transcription-coupled repair (TCR). However, in contrast to other reports, loss of TCR slightly increased the overall mutation rate, meaning that TCR is antimutagenic. There was no orientation bias in mutation frequencies among the stress-response genes that are regulated by RpoS or induced by DNA damage. Thus, biases in the locations of mutational targets can account for most, if not all, apparent biases in mutation frequencies between genes oriented head-on versus co-directional to replication. In addition, the data revealed a strong correlation of the frequency of base-pair substitutions with gene length, but no correlation with gene expression levels.
“…4 c). Because genes encoded on the lagging strand are mutated more frequently [ 37 ], an increased mutagenesis rate is likely less deleterious for sRNAs and/or potentially beneficial for the adaptation to the environmental changes. Fig.…”
Background
Targeted installation of designer chemical moieties on biopolymers provides an orthogonal means for their visualisation, manipulation and sequence analysis. Although high-throughput RNA sequencing is a widely used method for transcriptome analysis, certain steps, such as 3′ adapter ligation in strand-specific RNA sequencing, remain challenging due to structure- and sequence-related biases introduced by RNA ligases, leading to misrepresentation of particular RNA species. Here, we remedy this limitation by adapting two RNA 2′-O-methyltransferases from the Hen1 family for orthogonal chemo-enzymatic click tethering of a 3′ sequencing adapter that supports cDNA production by reverse transcription of the tagged RNA.
Results
We showed that the ssRNA-specific DmHen1 and dsRNA-specific AtHEN1 can be used to efficiently append an oligonucleotide adapter to the 3′ end of target RNA for sequencing library preparation. Using this new chemo-enzymatic approach, we identified miRNAs and prokaryotic small non-coding sRNAs in probiotic Lactobacillus casei BL23. We found that compared to a reference conventional RNA library preparation, methyltransferase-Directed Orthogonal Tagging and RNA sequencing, mDOT-seq, avoids misdetection of unspecific highly-structured RNA species, thus providing better accuracy in identifying the groups of transcripts analysed. Our results suggest that mDOT-seq has the potential to advance analysis of eukaryotic and prokaryotic ssRNAs.
Conclusions
Our findings provide a valuable resource for studies of the RNA-centred regulatory networks in Lactobacilli and pave the way to developing novel transcriptome and epitranscriptome profiling approaches in vitro and inside living cells. As RNA methyltransferases share the structure of the AdoMet-binding domain and several specific cofactor binding features, the basic principles of our approach could be easily translated to other AdoMet-dependent enzymes for the development of modification-specific RNA-seq techniques.
“…Genes, especially highly expressed, essential ones, are preferentially oriented such that they are transcribed, and accordingly translated, in the same direction as replication, presumably to avoid head-on collisions of transcription and replication (Brewer, 1988;Rocha, 2004;Schroeder et al, 2020). These biases thus orient Chi in the direction favoring repair of broken replication forks.…”
Section: Preferential Codon Usage Can Account For Chi's High Frequency In E Colimentioning
Bacteria face a challenge when DNA enters their cells by transformation, mating, or phage infection. Should they treat this DNA as an invasive foreigner and destroy it, or consider it one of their own and potentially benefit from incorporating new genes or alleles to gain useful functions? It is frequently stated that the short nucleotide sequence Chi (5’ GCTGGTGG 3’) recognized by RecBCD helicase-nuclease allows Escherichia coli to distinguish self (i.e., E. coli) DNA from non-self (i.e., any other) DNA and to destroy non-self DNA, and that Chi is “over-represented” in the E. coli genome. We show here that these dogmas are incorrect and apparently based on false assumptions. We note Chi’s wide-spread occurrence and activity in distantly related species. We illustrate multiple, highly non-random features of the genomes of coli and coliphage P1 that account for Chi’s high frequency and genomic position, leading us to propose that P1 selects for Chi’s enhancement of recombination, whereas E. coli selects for the preferred codons in Chi. We discuss other, substantiated mechanisms for self vs. non-self determination involving RecBCD and for RecBCD’s destruction of DNA that cannot recombine, whether foreign or domestic.
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