Nucleotide proofreading functions by nematode RAD51 paralogs facilitate optimal RAD51 filament function

The RAD51 recombinase assembles as helical nucleoprotein filaments on single-stranded DNA (ssDNA) and mediates invasion and strand exchange with homologous duplex DNA (dsDNA) during homologous recombination (HR), as well as protection and restart of stalled replication forks. Strand invasion by RAD51-ssDNA complexes depends on ATP binding. However, RAD51 can bind ssDNA in non-productive ADP-bound or nucleotide-free states, and ATP-RAD51-ssDNA complexes hydrolyse ATP over time. Here, we define unappreciated mechanisms by which the RAD51 paralog complex RFS-1/RIP-1 limits the accumulation of RAD-51-ssDNA complexes with unfavorable nucleotide content. We find RAD51 paralogs promote the turnover of ADP-bound RAD-51 from ssDNA, in striking contrast to their ability to stabilize productive ATP-bound RAD-51 nucleoprotein filaments. In addition, RFS-1/RIP-1 inhibits binding of nucleotide-free RAD-51 to ssDNA. We propose that ‘nucleotide proofreading’ activities of RAD51 paralogs co-operate to ensure the enrichment of active, ATP-bound RAD-51 filaments on ssDNA to promote HR.

Variation in Base Composition Underlies Functional and Evolutionary Divergence in Non-LTR Retrotransposons

ABSTRACTBackgroundNon-LTR retrotransposons often exhibit base composition that is markedly different from the nucleotide content of their host’s gene. For instance, the mammalian L1 element is AT-rich with a strong A bias on the positive strand, which results in a reduced transcription. It is plausible that the A-richness of mammalian L1 is a self-regulatory mechanism reflecting a trade-off between transposition efficiency and the deleterious effect of L1 on its host. We examined if the A-richness of L1 is a general feature of non-LTR retrotransposons or if different clades of elements have evolved different nucleotide content. We also investigated if elements belonging to the same clade evolved towards different base composition in different genomes or if elements from the same clades evolved towards similar base composition in the same genome.ResultsWe found that non-LTR retrotransposons differ in base composition among clades within the same host but also that elements belonging to the same clade differ in base composition among hosts. We showed that nucleotide content remains constant within the same host over extended period of evolutionary time, despite mutational patterns that should drive nucleotide content away from the observed base composition.ConclusionsOur results suggest that base composition is evolving under selection and may be reflective of the long-term co-evolution between non-LTR retrotransposons and their host. Finally, the coexistence of elements with drastically different base composition suggests that these elements may be using different strategies to persist and multiply in the genome of their host.

The most primitive extant ancestor of organisms and discovery of definitive evolutionary equations

Organisms are classified into three domains, Prokaryota, Archaea, and Eukaryota, and their evolutionary divergence has been characterized based on morphological and molecular features using rationale based on Darwin’s theory of natural selection. However, universal rules that govern genome evolution have not been identified. Here, a simple, innovative approach has been developed to evaluate biological evolution initiating the origin of life: whole genomes were divided into several fragments, and then differences in normalized nucleotide content between nucleotide pairs were compared. Based on nucleotide content structures, Monosiga brevicollis mitochondria may be the most primitive extant ancestor of the species examined here. The two normalized nucleotide contents are universally expressed by a linear regression line, (X − Y)/(X + Y) = a (X − Y) + b, where X and Y are nucleotide contents and (a) and (b) are constants. The value of (G + C), (G + A), (G + T), (C + A), (C + T) and (A + T) was ~0.5. Plotting (X − Y)/(X + Y) against X/Y showed a logarithmic function (X − Y)/(X + Y) = a ln X/Y + b, where (a) and (b) are constant. Nucleotide content changes are expressed by a definitive equation, (X − Y) ≈ 0.25 ln(X/Y).

Biases in genome reconstruction from metagenomic data

Background: Technological advances in sequencing, assembly and segregation of resulting contigs into species-specific bins has enabled the reconstruction of individual genomes from environmental metagenomic data sets. Though a powerful technique, it is shadowed by an inability to truly determine whether assembly and binning techniques are accurate, specific, and sensitive due to a lack of complete reference genome sequences against which to check the data. Errors in genome reconstruction, such as missing or mis-attributed activities, can have a detrimental effect on downstream metabolic and ecological modeling, and thus it is important to assess the accuracy of the process. Methods: We compared genomes reconstructed from metagenomic data to complete genome sequences of 10 organisms isolated from the same community to identify regions not captured by typical binning techniques. The nucleotide content, as %G+C and tetranucleotide frequencies, and sequence redundancy within both the genome and across the metagenome were determined for both the captured and uncaptured regions. This direct comparison allowed us to evaluate the efficacy of nucleotide composition and coverage profiles as elements of binning protocols and look for biases in sequence characteristics and gene content in regions missing from the reconstructions. Results: We found that repeated sequences were frequently missed in the reconstruction process as were short sequences with variant nucleotide composition. Genes encoded on the missing regions were strongly biased towards ribosomal RNAs, transfer RNAs, mobile element functions and genes of unknown function. Conclusions: Our observation of increased mis-binning of short regions, especially those with variant nucleotide content, and repeated regions implies that factors which affect assembly efficiency also impact binning accuracy. To a large extent, mis-binned regions appear to derive from mobile elements. Our results support genome reconstruction as a robust process, and suggest that reconstructions determined to be >90% complete are likely to effectively represent organismal function.

Biases in genome reconstruction from metagenomic data

Background: Technological advances in sequencing, assembly and segregation of resulting contigs into species-specific bins has enabled the reconstruction of individual genomes from environmental metagenomic data sets. Though a powerful technique, it is shadowed by an inability to truly determine whether assembly and binning techniques are accurate, specific, and sensitive due to a lack of complete reference genome sequences against which to check the data. Errors in genome reconstruction, such as missing or mis-attributed activities, can have a detrimental effect on downstream metabolic and ecological modeling, and thus it is important to assess the accuracy of the process. Methods: We compared genomes reconstructed from metagenomic data to complete genome sequences of 10 organisms isolated from the same community to identify regions not captured by typical binning techniques. The nucleotide content, as %G+C and tetranucleotide frequencies, and sequence redundancy within both the genome and across the metagenome were determined for both the captured and uncaptured regions. This direct comparison allowed us to evaluate the efficacy of nucleotide composition and coverage profiles as elements of binning protocols and look for biases in sequence characteristics and gene content in regions missing from the reconstructions. Results: We found that repeated sequences were frequently missed in the reconstruction process as were short sequences with variant nucleotide composition. Genes encoded on the missing regions were strongly biased towards ribosomal RNAs, transfer RNAs, mobile element functions and genes of unknown function. Conclusions: Our observation of increased mis-binning of short regions, especially those with variant nucleotide content, and repeated regions implies that factors which affect assembly efficiency also impact binning accuracy. To a large extent, mis-binned regions appear to derive from mobile elements. Our results support genome reconstruction as a robust process, and suggest that reconstructions determined to be >90% complete are likely to effectively represent organismal function.