The Retrohoming of Linear Group II Intron RNAs in Drosophila melanogaster Occurs by Both DNA Ligase 4–Dependent and –Independent Mechanisms

Mobile group II introns are bacterial retrotransposons that are thought to have invaded early eukaryotes and evolved into introns and retroelements in higher organisms. In bacteria, group II introns typically retrohome via full reverse splicing of an excised intron lariat RNA into a DNA site, where it is reverse transcribed by the intron-encoded protein. Recently, we showed that linear group II intron RNAs, which can result from hydrolytic splicing or debranching of lariat RNAs, can retrohome in eukaryotes by performing only the first step of reverse splicing, ligating their 3′ end to the downstream DNA exon. Reverse transcription then yields an intron cDNA, whose free end is linked to the upstream DNA exon by an error-prone process that yields junctions similar to those formed by non-homologous end joining (NHEJ). Here, by using Drosophila melanogaster NHEJ mutants, we show that linear intron RNA retrohoming occurs by major Lig4-dependent and minor Lig4-independent mechanisms, which appear to be related to classical and alternate NHEJ, respectively. The DNA repair polymerase θ plays a crucial role in both pathways. Surprisingly, however, mutations in Ku70, which functions in capping chromosome ends during NHEJ, have only moderate, possibly indirect effects, suggesting that both Lig4 and the alternate end-joining ligase act in some retrohoming events independently of Ku. Another potential Lig4-independent mechanism, reverse transcriptase template switching from the intron RNA to the upstream exon DNA, occurs in vitro, but gives junctions differing from the majority in vivo. Our results show that group II introns can utilize cellular NHEJ enzymes for retromobility in higher organisms, possibly exploiting mechanisms that contribute to retrotransposition and mitigate DNA damage by resident retrotransposons. Additionally, our results reveal novel activities of group II intron reverse transcriptases, with implications for retrohoming mechanisms and potential biotechnological applications

Heterochromatic Genome Stability Requires Regulators of Histone H3 K9 Methylation

Heterochromatin contains many repetitive DNA elements and few protein-encoding genes, yet it is essential for chromosome organization and inheritance. Here, we show that Drosophila that lack the Su(var)3-9 H3K9 methyltransferase display significantly elevated frequencies of spontaneous DNA damage in heterochromatin, in both somatic and germ-line cells. Accumulated DNA damage in these mutants correlates with chromosomal defects, such as translocations and loss of heterozygosity. DNA repair and mitotic checkpoints are also activated in mutant animals and are required for their viability. Similar effects of lower magnitude were observed in animals that lack the RNA interference pathway component Dcr2. These results suggest that the H3K9 methylation and RNAi pathways ensure heterochromatin stability

Competitive Repair by Naturally Dispersed Repetitive DNA during Non-Allelic Homologous Recombination

Genome rearrangements often result from non-allelic homologous recombination (NAHR) between repetitive DNA elements dispersed throughout the genome. Here we systematically analyze NAHR between Ty retrotransposons using a genome-wide approach that exploits unique features of Saccharomyces cerevisiae purebred and Saccharomyces cerevisiae/Saccharomyces bayanus hybrid diploids. We find that DNA double-strand breaks (DSBs) induce NAHR–dependent rearrangements using Ty elements located 12 to 48 kilobases distal to the break site. This break-distal recombination (BDR) occurs frequently, even when allelic recombination can repair the break using the homolog. Robust BDR–dependent NAHR demonstrates that sequences very distal to DSBs can effectively compete with proximal sequences for repair of the break. In addition, our analysis of NAHR partner choice between Ty repeats shows that intrachromosomal Ty partners are preferred despite the abundance of potential interchromosomal Ty partners that share higher sequence identity. This competitive advantage of intrachromosomal Tys results from the relative efficiencies of different NAHR repair pathways. Finally, NAHR generates deleterious rearrangements more frequently when DSBs occur outside rather than within a Ty repeat. These findings yield insights into mechanisms of repeat-mediated genome rearrangements associated with evolution and cancer

The Science Performance of JWST as Characterized in Commissioning

This paper characterizes the actual science performance of the James Webb Space Telescope (JWST), as determined from the six month commissioning period. We summarize the performance of the spacecraft, telescope, science instruments, and ground system, with an emphasis on differences from pre-launch expectations. Commissioning has made clear that JWST is fully capable of achieving the discoveries for which it was built. Moreover, almost across the board, the science performance of JWST is better than expected; in most cases, JWST will go deeper faster than expected. The telescope and instrument suite have demonstrated the sensitivity, stability, image quality, and spectral range that are necessary to transform our understanding of the cosmos through observations spanning from near-earth asteroids to the most distant galaxies

The Science Performance of JWST as Characterized in Commissioning

This paper characterizes the actual science performance of the James Webb Space Telescope (JWST), as determined from the six month commissioning period. We summarize the performance of the spacecraft, telescope, science instruments, and ground system, with an emphasis on differences from pre-launch expectations. Commissioning has made clear that JWST is fully capable of achieving the discoveries for which it was built. Moreover, almost across the board, the science performance of JWST is better than expected; in most cases, JWST will go deeper faster than expected. The telescope and instrument suite have demonstrated the sensitivity, stability, image quality, and spectral range that are necessary to transform our understanding of the cosmos through observations spanning from near-earth asteroids to the most distant galaxies.Comment: 5th version as accepted to PASP; 31 pages, 18 figures; https://iopscience.iop.org/article/10.1088/1538-3873/acb29

Processing of joint molecule intermediates by structure-selective endonucleases during homologous recombination in eukaryotes

Homologous recombination is required for maintaining genomic integrity by functioning in high-fidelity repair of DNA double-strand breaks and other complex lesions, replication fork support, and meiotic chromosome segregation. Joint DNA molecules are key intermediates in recombination and their differential processing determines whether the genetic outcome is a crossover or non-crossover event. The Holliday model of recombination highlights the resolution of four-way DNA joint molecules, termed Holliday junctions, and the bacterial Holliday junction resolvase RuvC set the paradigm for the mechanism of crossover formation. In eukaryotes, much effort has been invested in identifying the eukaryotic equivalent of bacterial RuvC, leading to the discovery of a number of DNA endonucleases, including Mus81–Mms4/EME1, Slx1–Slx4/BTBD12/MUS312, XPF–ERCC1, and Yen1/GEN1. These nucleases exert different selectivity for various DNA joint molecules, including Holliday junctions. Their mutant phenotypes and distinct species-specific characteristics expose a surprisingly complex system of joint molecule processing. In an attempt to reconcile the biochemical and genetic data, we propose that nicked junctions constitute important in vivo recombination intermediates whose processing determines the efficiency and outcome (crossover/non-crossover) of homologous recombination

Type I Repressors of P Element Mobility

We describe here a family of P elements that we refer to as type I repressors. These elements are identified by their repressor functions and their lack of any deletion within the first two-thirds of the canonical P sequence. Elements belonging to this repressor class were isolated from P strains and were made in vitro. We found that type I repressor elements could strongly repress both a cytotype-dependent allele and P element mobility in somatic and germline tissues. These effects wer very dependent on genomic position. Moreover, we observed that an element's ability to repress in one assay positively correlated with its ability to repress in either of the other two assays. The type I family of repressor elements includes both autonomous P elements and those lacking exon 3 of the P element. Fine structure deletion mapping showed that the minimal 3' boundary of a functional type I element lies between nucleotide position 1950 and 1956. None of 12 elements examined with more extreme deletions extending into exon 2 made repressor. We conclude that the type I repressors form a structurally distinct group that does not include more extensively deleted repressor elements such as the KP element described previously