Biology subjects

Crown, K. N.

Publications and source records attributed to Crown, K. N..

3 recordsLinked to original sources

bioRxiv2023

Single-fly assemblies fill major phylogenomic gaps across the Drosophilidae Tree of Life

Long-read sequencing is driving rapid progress in genome assembly across all major groups of life, including species of the family Drosophilidae, a longtime model system for genetics, genomics, and evolution. We previously developed a cost-effective hybrid Oxford Nanopore (ONT) long-read and Illumina short-read sequencing approach and used it to assemble 101 drosophilid genomes from laboratory cultures, greatly increasing the number of genome assemblies for this taxonomic group. The next major challenge is to address the laboratory culture bias in taxon sampling by sequencing genomes of species that cannot easily be reared in the lab. Here, we build upon our previous methods to perform amplification-free ONT sequencing of single wild flies obtained either directly from the field or from ethanol-preserved specimens in museum collections, greatly improving the representation of lesser studied drosophilid taxa in whole-genome data. Using Illumina Novaseq X Plus and ONT P2 sequencers with R10.4.1 chemistry, we set a new benchmark for inexpensive hybrid genome assembly at US $150 per genome while assembling genomes from as little as 35 ng of genomic DNA from a single fly. We present 183 new genome assemblies for 179 species as a resource for drosophilid systematics, phylogenetics, and comparative genomics. Of these genomes, 62 are from pooled lab strains and 121 from single adult flies. Despite the sample limitations of working with small insects, most single-fly diploid assemblies are comparable in contiguity (>1Mb contig N50), completeness (>98% complete dipteran BUSCOs), and accuracy (>QV40 genome-wide with ONT R10.4.1) to assemblies from inbred lines. We present a well-resolved multi-locus phylogeny for 360 drosophilid and 4 outgroup species encompassing all publicly available (as of August 2023) genomes for this group. Finally, we present a Progressive Cactus whole-genome, reference-free alignment built from a subset of 298 suitably high-quality drosophilid genomes. The new assemblies and alignment, along with updated laboratory protocols and computational pipelines, are released as an open resource and as a tool for studying evolution at the scale of an entire insect family.

evolutionary biology↗

bioRxiv2022

Heterozygous Inversion Breakpoints Suppress Meiotic Crossovers by Altering Recombination Repair Outcomes

Heterozygous chromosome inversions suppress meiotic crossover (CO) formation within an inversion, potentially because they lead to gross chromosome rearrangements that produce inviable gametes. Interestingly, COs are also severely reduced in regions nearby but outside of inversion breakpoints even though COs in these regions do not result in rearrangements. Our mechanistic understanding of why COs are suppressed outside of inversion breakpoints is limited by a lack of data on the frequency of noncrossover gene conversions (NCOGCs) in these regions. To address this critical gap, we mapped the location and frequency of rare CO and NCOGC events that occurred outside of the dl-49 chrX inversion in D. melanogaster. We created full-sibling wildtype and inversion stocks and recovered COs and NCOGCs in the syntenic regions of both stocks, allowing us to directly compare rates and distributions of recombination events. We show that COs are completely suppressed within 500 kb of inversion breakpoints, are severely reduced within 2 Mb of an inversion breakpoint, and increase above wildtype levels 2-4 Mb from the breakpoint. We find that NCOGCs occur evenly throughout the chromosome and, importantly, occur at wildtype levels near inversion breakpoints. We propose a model in which COs are suppressed by inversion breakpoints in a distance-dependent manner through mechanisms that influence DNA double-strand break repair outcome but not double-strand break location or frequency. We suggest that subtle changes in the synaptonemal complex and chromosome pairing might lead to unstable interhomolog interactions during recombination that permits NCOGC formation but not CO formation.

genetics↗

bioRxiv2020

DNA polymerase theta suppresses mitotic crossing over

Polymerase theta-mediated end joining (TMEJ) is a chromosome break repair pathway that is able to rescue the lethality associated with the loss of proteins involved in early steps in homologous recombination (e.g., BRCA1/2). This is due to the ability of polymerase theta (Pol {theta}) to use resected, 3 single stranded DNA tails to repair chromosome breaks. These resected DNA tails are also the starting substrate for homologous recombination. However, it remains unknown if TMEJ can compensate for the loss of proteins involved in more downstream steps during homologous recombination. Here we expand the number of homologous recombination proteins synthetic lethal with Pol {theta} to the Holliday junction resolvases SLX4 and GEN1. SLX4 and GEN1 are required for viability in the absence of Pol {theta} in Drosophila melanogaster, and lack of all three proteins results in very high levels of apoptosis. We observe that flies deficient in Pol {theta} and SLX4 are extremely sensitive to DNA damaging agents, and mammalian cells require either Pol {theta} or SLX4 to survive. Our results suggest that TMEJ and Holliday junction formation/resolution share a common DNA substrate, likely a homologous recombination intermediate, that when left unrepaired leads to cell death. One major consequence of Holliday junction resolution by SLX4 and GEN1 is cancer-causing loss of heterozygosity due to mitotic crossing over. We measured mitotic crossovers in flies after a Cas9-induced chromosome break, and observed that this mutagenic form of repair is increased in the absence of Pol {theta}. This demonstrates that TMEJ can function upstream of the Holiday junction resolvases to protect cells from loss of heterozygosity. Our work argues that Pol {theta} can thus compensate for the loss of the Holliday junction resolvases by utilizing homologous recombination intermediates, suppressing mitotic crossing over and preserving the genomic stability of cells. Author summaryChromosome breaks are a common threat to the stability of DNA. Mutations in genes involved in the early steps of homologous recombination (BRCA1 and BRCA2), a mostly error-free chromosome break repair pathway, lead to hereditary breast cancer. Cells lacking BRCA1 and BRCA2 rely on DNA polymerase theta, a key protein for a more error-prone pathway, for survival. Using fruit flies and mammalian cells, we have shown that mutations in genes involved in later steps of homologous recombination (SLX4 and GEN1) also make cells reliant on polymerase theta. Moreover, we have shown that polymerase theta acts upstream of a type of homologous recombination that is error-prone and depends on SLX4 and GEN1. This form of homologous recombination, termed Holliday junction resolution, creates mitotic crossovers, which can lead to loss of heterozygosity and cancer. Our results expand the cellular contexts that make cells depend on polymerase theta for survival, and the substrates that this protein can use to repair chromosome breaks.

genetics↗