Chromosome breaks in breast cancers occur near herpes tumor virus sequences

This work finds viral DNA associates with most chromosome breaks in breast cancer and provides a mechanism for why this is so. Nearly 2000 breast cancers were compared to known Epstein Barr virus (EBV) variant cancers using publicly available data. Breast cancer breakpoints on all chromosomes cluster around the same positions as in nasopharyngeal cancers (NPCs), cancers 100 per cent associated with EBV variants. Breakpoints also gather at the same differentially methylated regions. Breast cancer further has an EBV methylation signature shared with other cancers that inactivates complement. Another known EBV cancer (Burkitt lymphoma) has distinctive MYC gene breakpoints surrounded by EBV like DNA. EBV like DNA consistently surrounds breast cancer breakpoints, which are often near known EBV binding sites. EBV explains why a break in a chromosome does not simply reconnect in breakage fusion bridge models, but instead destabilizes the entire genome. This work does not prove EBV variants cause breast cancer, but establishes links to high risk chromosome breaks and other changes.

Sequencing Micronuclei Reveals the Landscape of Chromosomal Instability

Genome instability (GIN) is a main contributing factor to congenital and somatic diseases, but its sporadic occurrence in individual cell cycles makes it difficult to study mechanistically. One profound manifestation of GIN is the formation of micronuclei (MN), the engulfment of chromosomes or chromosome fragments in their own nuclear structures separate from the main nucleus. Here, we developed MN-seq, an approach for sequencing the DNA contained within micronuclei. We applied MN-seq to mice with mutations in Mcm4 and Rad9a, which disrupt DNA replication, repair, and damage responses. Data analysis and simulations show that centromere presence, fragment length, and a heterogenous landscape of chromosomal fragility all contribute to the patterns of DNA present within MN. In particular, we show that long genes, but also gene-poor regions, are associated with chromosome breaks that lead to the enrichment of particular genomic sequences in MN, in a genetic background-specific manner. Finally, we introduce single-cell micronucleus sequencing (scMN-seq), an approach to sequence the DNA present in MN of individual cells. Together, sequencing micronuclei provides a systematic approach for studying GIN and reveals novel molecular associations with chromosome breakage and segregation.

Unscheduled DNA replication in G1 causes genome instability through head-to-tail replication fork collisions

DNA replicates once per cell cycle. Interfering with the regulation of DNA replication initiation generates genome instability through over-replication and has been linked to early stages of cancer development. Here, we engineered genetic systems in budding yeast to induce unscheduled replication in the G1-phase of the cell cycle. Unscheduled G1 replication initiated at canonical S-phase origins across the genome. We quantified differences in replisomes in G1- and S-phase and identified firing factors, polymerase α, and histone supply as factors that limit replication outside S-phase. G1 replication per se did not trigger cellular checkpoints. Subsequent replication during S-phase, however, resulted in over-replication and led to chromosome breaks via head-to-tail replication fork collisions that are marked by chromosome-wide, strand-biased occurrence of RPA-bound single-stranded DNA. Low-level, sporadic induction of G1 replication induced an identical response, indicating findings from synthetic systems are applicable to naturally occurring scenarios of unscheduled replication initiation by G1/S deregulation.

In BRCA1 and BRCA2 Breast Cancers, Chromosome Breaks Occur near Herpes Tumor Virus Sequences

Inherited mutations in BRCA1 and BRCA2 genes increase risks for breast, ovarian, and other cancers. Both genes encode proteins for accurately repairing chromosome breaks. If mutations inactivate this function, broken chromosomes may not be restored correctly, allowing breaks to persist or rearrange chromosomes. These abnormalities are potentially catastrophic events that can originate from viral infections. I used bioinformatic analyses of publicly available breast cancer patient data to show that the distribution of chromosome breaks in hereditary breast cancers differs markedly from sporadic breast cancers. Then I tested hereditary breast cancer sequence data around chromosome breaks for DNA similarity to all known viruses. Human DNA flanking breakpoints usually had decisive matches to Epstein-Barr virus (EBV / HHV4) tumor variants HKHD40 and HKNPC60. Many breakpoints were near EBV genome anchor sites, human EBV tumor-like sequences, EBV-associated epigenetic marks, and some fragile sites. On chromosomes 2 and 12, sequences near EBV genome anchor sites accounted for 90% and 88% of breakpoints (p<0.0001), respectively. On chromosome 4, 51/52 inter-chromosomal breakpoints were close to EBV-like sequences in 19 hereditary breast cancers. In contrast, 19 sporadic breast cancers only had 12 interchromosomal breakpoint regions on chromosome 4 near EBV-like sequences. On various other chromosomes, five EBV genome anchor sites were near hereditary breast cancer breakpoints at precisely defined, disparate gene or LINE locations. Independent evidence further implicating EBV in hereditary breast cancer breakpoints is that 25 breast cancer break positions are within 1.25% of breakpoints in model EBV cancers. In addition to BRCA1 or BRCA2 mutations, all the hereditary breast cancers had mutated genes essential for immune responses. This compromise facilitates reactivation of herpes viruses which produce nucleases capable of breaking chromosomes. EBV also causes other deleterious effects: anchored EBV episomes can interfere with normal replication and obstruct DNA break repairs; even very early infection causes massive transcription changes. The results, therefore, imply proactive treatment and prevention of herpes viral infections may prevent some chromosome breaks and benefit BRCA mutation carriers.

In BRCA1 and BRCA2 Breast Cancers, Chromosome Breaks Occur Near Herpes Tumor Virus Sequences

Inherited mutations in BRCA1 and BRCA2 genes increase risks for breast, ovarian, and other cancers. Both genes encode proteins for accurately repairing chromosome breaks. If mutations inactivate this function, chromosome fragments may not be restored correctly. Resulting chromosome rearrangements can become critical breast cancer drivers. Because I had data from thousands of cancer structural alterations that matched viral infections, I wondered whether infections contribute to chromosome breaks and rearrangements in hereditary breast cancers. There are currently no interventions to prevent chromosome breaks because they are thought to be unavoidable. However, if chromosome breaks come from infections, they can be treated or prevented. I used bioinformatic analyses to test publicly available breast cancer sequence data around chromosome breaks for DNA similarity to all known viruses. Human DNA flanking breakpoints usually had the strongest matches to Epstein-Barr virus (EBV) tumor variants HKHD40 and HKNPC60. Many breakpoints were near sites that anchor EBV genomes, human EBV tumor-like sequences, EBV-associated epigenetic marks, and fragile sites. On chromosome 2, sequences near EBV genome anchor sites accounted for 90% of breakpoints (p<0.0001). On chromosome 4, 51/52 inter-chromosomal breakpoints were close to EBV-like sequences. Five EBV genome anchor sites were near breast cancer breakpoints at precisely defined, disparate gene or LINE locations. Breakpoint flanking regions resembled known EBV-cancers. Twenty-five breakpoints in breast cancers were within 1.25% of EBV cancer breakpoints. In addition to BRCA1 or BRCA2 mutations, all the breast cancers had mutated genes essential for immune responses. Because of this immune compromise, herpes viruses can activate and produce nucleases that break chromosomes. Alternatively, anchored viral episomes can obstruct break repairs, whatever the cause. The results, therefore, imply proactive treatment and prevention of herpes viral infections may prevent some chromosome breaks and benefit BRCA mutation carriers.

In BRCA1 and BRCA2 breast cancers, chromosome breaks occur near herpes tumor virus sequences

Inherited mutations in BRCA1 and BRCA2 genes increase risks for breast, ovarian, and other cancers. Both genes encode proteins for accurately repairing chromosome breaks. If mutations inactivate this function, broken chromosome fragments get lost or reattach indiscriminately. These mistakes are characteristic of hereditary breast cancer. We tested the hypothesis that mistakes in reattaching broken chromosomes preferentially occur near viral sequences on human chromosomes. We tested millions of DNA bases around breast cancer breakpoints for similarities to all known viral DNA. DNA around breakpoints often closely matched the Epstein-Barr virus (EBV) tumor variants HKHD40 and HKNPC60. Almost all breakpoints were near EBV anchor sites, EBV tumor variant homologies, and EBV-associated regulatory marks. On chromosome 2, EBV binding sites accounted for 90% of breakpoints (p<0.0001). On chromosome 4, 51/52 inter-chromosomal breakpoints were close to EBV variant sequences. Five viral anchor sites at critical genes were near breast cancer breakpoints. Twenty-five breast cancer breakpoints were within 1.25% of breakpoints in model EBV cancers. EBV-like sequence patterns around breast cancer breakpoints resemble gene fusion breakpoints in model EBV cancers. All BRCA1 and BRCA2 breast cancers had mutated genes essential for immune responses. Because of this immune compromise, herpes viruses can attach and produce nucleases that break chromosomes. Alternatively, anchored viruses can retard break repairs, whatever the causes. The results imply proactive treatment and prevention of herpes viral infections may benefit BRCA mutation carriers.

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 θ) 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 θ to the Holliday junction resolvases SLX4 and GEN1. SLX4 and GEN1 are required for viability in the absence of Pol θ in Drosophila melanogaster, and lack of all three proteins results in very high levels of apoptosis. We observe that flies deficient in Pol θ and SLX4 are extremely sensitive to DNA damaging agents, and mammalian cells require either Pol θ 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 θ. This demonstrates that TMEJ can function upstream of the Holiday junction resolvases to protect cells from loss of heterozygosity. Our work argues that Pol θ 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.

Rare instances of haploid inducer DNA in potato dihaploids and ploidy-dependent genome instability

In cultivated tetraploid potato, reduction to diploidy (dihaploidy) allows hybridization to diploid germplasm, introgression breeding, and may facilitate the production of inbreds. Pollination with haploid inducers yields maternal dihaploids, as well as triploid and tetraploid hybrids. It is not known if dihaploids result from parthenogenesis, entailing development of embryos from unfertilized eggs, or genome elimination, entailing missegregation and loss of paternal chromosomes. A sign of genome elimination is the occasional persistence of haploid inducer DNA in some of the dihaploids. We characterized the genomes of 1,001 putative dihaploids and 134 hybrids produced by pollinating tetraploid clones with three haploid inducers, IVP35, IVP101, and PL4. We detected inheritance of full or partial chromosomes from the haploid inducer parent in 0.87% of the overall dihaploid progeny, irrespective of the combination of parental genotypes. Chromosomal breaks commonly affected the paternal genome in the dihaploid and tetraploid progeny, but not in the triploid progeny. Residual haploid inducer DNA is consistent with genome elimination as the mechanism of haploid induction. Further, the fact that paternal chromosome breaks are specific to dihaploids and tetraploid progeny suggests that they may be specific to 2x sperms, and supports the hypothesis that 2x sperms facilitate genome elimination.

Doxorubicin-Induced Translocation of mtDNA into the Nuclear Genome of Human Lymphocytes Detected Using a Molecular-Cytogenetic Approach

Translocation of mtDNA in the nuclear genome is an ongoing process that contributes to the development of pathological conditions in humans. However, the causal factors of this biological phenomenon in human cells are poorly studied. Here we analyzed mtDNA insertions in the nuclear genome of human lymphocytes after in vitro treatment with doxorubicin (DOX) using a fluorescence in situ hybridization (FISH) technique. The number of mtDNA insertions positively correlated with the number of DOX-induced micronuclei, suggesting that DOX-induced chromosome breaks contribute to insertion events. Analysis of the odds ratios (OR) revealed that DOX at concentrations of 0.025 and 0.035 µg/mL significantly increases the rate of mtDNA insertions (OR: 3.53 (95% CI: 1.42–8.76, p < 0.05) and 3.02 (95% CI: 1.19–7.62, p < 0.05), respectively). Analysis of the distribution of mtDNA insertions in the genome revealed that DOX-induced mtDNA insertions are more frequent in larger chromosomes, which are more prone to the damaging action of DOX. Overall, our data suggest that DOX-induced chromosome damage can be a causal factor for insertions of mtDNA in the nuclear genome of human lymphocytes. It can be assumed that the impact of a large number of external and internal mutagenic factors contributes significantly to the origin and amount of mtDNA in nuclear genomes.

The Greatwall kinase safeguards the genome integrity by affecting the kinome activity in mitosis

Progression through mitosis is balanced by the timely regulation of phosphorylation and dephosphorylation events ensuring the correct segregation of chromosomes before cytokinesis. This balance is regulated by the opposing actions of CDK1 and PP2A, as well as the Greatwall kinase/MASTL. MASTL is commonly overexpressed in cancer, which makes it a potential therapeutic anticancer target. Loss of Mastl induces multiple chromosomal errors that lead to the accumulation of micronuclei and multilobulated cells in mitosis. Our analyses revealed that loss of Mastl leads to chromosome breaks and abnormalities impairing correct segregation. Phospho-proteomic data for Mastl knockout cells revealed alterations in proteins implicated in multiple processes during mitosis including double-strand DNA damage repair. In silico prediction of the kinases with affected activity unveiled NEK2 to be regulated in the absence of Mastl. We uncovered that, RAD51AP1, involved in regulation of homologous recombination, is phosphorylated by NEK2 and CDK1 but also efficiently dephosphorylated by PP2A/B55. Our results suggest that MastlKO disturbs the equilibrium of the mitotic phosphoproteome that leads to the disruption of DNA damage repair and triggers an accumulation of chromosome breaks even in noncancerous cells.