Dynamin impacts homology-directed repair and breast cancer response to chemotherapy.

After the initial responsiveness of triple-negative breast cancers (TNBCs) to chemotherapy, they often recur as chemotherapy-resistant tumors, and this has been associated with upregulated homology-directed repair (HDR). Thus, inhibitors of HDR could be a useful adjunct to chemotherapy treatment of these cancers. We performed a high-throughput chemical screen for inhibitors of HDR from which we obtained a number of hits that disrupted microtubule dynamics. We postulated that high levels of the target molecules of our screen in tumors would correlate with poor chemotherapy response. We found that inhibition or knockdown of dynamin 2 (DNM2), known for its role in endocytic cell trafficking and microtubule dynamics, impaired HDR and improved response to chemotherapy of cells and of tumors in mice. In a retrospective analysis, levels of DNM2 at the time of treatment strongly predicted chemotherapy outcome for estrogen receptor-negative and especially for TNBC patients. We propose that DNM2-associated DNA repair enzyme trafficking is important for HDR efficiency and is a powerful predictor of sensitivity to breast cancer chemotherapy and an important target for therapy.

Inhibiting homologous recombination for cancer therapy.

We review the rationale for seeking inhibitors of homologous recombination (HR) repair for use in cancer therapy. Cells use HR as one way to repair DNA double-strand breaks that arise directly from treatments such as radiotherapy, or indirectly during replication when forks encounter other damage. HR occurs during the S and G 2 phases of the cell cycle and is therefore more significant in dividing cancer cells than in non-dividing cells of healthy tissue, giving a potential therapeutic advantage to inhibiting the process. Also, some tumors consist of cells that are defective in other DNA repair pathways, and such cells may be sensitive to HR repair inhibitors because of synthetic lethality, in which blocking two alternative pathways that a cell can use to reach a needed end-point has a much bigger impact than blocking either pathway alone. We review strategies for identifying HR inhibitors and discuss current progress.

Deficiency in mammalian histone H2B ubiquitin ligase Bre1 (Rnf20/Rnf40) leads to replication stress and chromosomal instability.

Mammalian Bre1 complexes (BRE1A/B (RNF20/40) in humans and Bre1a/b (Rnf20/40) in mice) function similarly to their yeast homolog Bre1 as ubiquitin ligases in monoubiquitination of histone H2B. This ubiquitination facilitates methylation of histone H3 at K4 and K79, and accounts for the roles of Bre1 and its homologs in transcriptional regulation. Recent studies by others suggested that Bre1 acts as a tumor suppressor, augmenting expression of select tumor suppressor genes and suppressing select oncogenes. In this study, we present an additional mechanism of tumor suppression by Bre1 through maintenance of genomic stability. We track the evolution of genomic instability in Bre1-deficient cells from replication-associated double-strand breaks (DSB) to specific genomic rearrangements that explain a rapid increase in DNA content and trigger breakage-fusion-bridge cycles. We show that aberrant RNA-DNA structures (R-loops) constitute a significant source of DSBs in Bre1-deficient cells. Combined with a previously reported defect in homologous recombination, generation of R-loops is a likely initiator of replication stress and genomic instability in Bre1-deficient cells. We propose that genomic instability triggered by Bre1 deficiency may be an important early step that precedes acquisition of an invasive phenotype, as we find decreased levels of BRE1A/B and dimethylated H3K79 in testicular seminoma and in the premalignant lesion in situ carcinoma.

Deficiency in Bre1 impairs homologous recombination repair and cell cycle checkpoint response to radiation damage in mammalian cells.

The pathway involving Bre1-dependent monoubiquitination of histone H2B lysine 123, which leads to Dot1-dependent methylation of histone H3 lysine 79 (H3K79me2), has been implicated in survival after exposure to ionizing radiation in Saccharomyces cerevisiae. We found that depletion of mammalian homologs of Bre1 compromises the response to ionizing radiation, leading to increased radiosensitivity and a G(2)/M checkpoint defect. The deficiency in Bre1a/b function was also associated with increased sensitivity to crosslinking drugs and defective formation of Rad51 foci in mouse cells, suggesting a defect in homologous recombinational repair analogous to that seen in Saccharomyces. In budding yeast, H3K79me2 is important for the recruitment of the checkpoint signaling protein Rad9 to sites of double-strand breaks (DSBs). However, in mammalian cells, 53BP1 (the Rad9 ortholog) in addition to H3K79me2 recognizes a different residue, H4K20me2, and some studies argue that it is H4K20me2 and not H3K79me2 that is the preferred target for 53BP1. We show here that depletion of Bre1b specifically reduced dimethylation of H3K79 without affecting dimethylation of H4K20. Thus our data suggest that the observed defects in the radiation response of Bre1a/b-deficient cells are associated with reduced H3K79me2 and not with H4K20me2.

The role of RAD6 in recombinational repair, checkpoints and meiosis via histone modification.

The Rad6 ubiquitin-conjugating enzyme in Saccharomyces cerevisiae is known to interact with three separate ubiquitin ligase proteins (Ubr1, Rad18, and Bre1) specific to different targets. The Rad6/Rad18 complex is central to translesion synthesis and the family of DNA transactions known as post-replication repair (PRR). A less well-known aspect of Rad6-mediated DNA repair, however, involves its function with Bre1 in mono-ubiquitinating the histone H2B residue lysine 123. Here, we review how this ubiquitination impacts histone H3 methylation, and how this in turn impacts the DNA damage response. In S. cerevisiae this pathway is required for checkpoint activation in G1, and contributes to DNA repair via the homologous recombination pathway (HRR) in G2 cells. Thus, RAD6 clearly plays a role in HRR in addition to its central role in PRR. We also summarize what is known about related repair pathways in other eukaryotes, including mammals. Recent literature emphasizes the role of methylated histones in S. cerevisiae, Schizosaccharomyces pombe and mammals in attracting the related DNA damage checkpoint proteins Rad9, Crb2 and 53BP1, respectively, to chromatin at the sites of DNA double-strand breaks. However, the specific histone modification pathways involved diverge in these different eukaryotes.

The RAD6/BRE1 histone modification pathway in Saccharomyces confers radiation resistance through a RAD51-dependent process that is independent of RAD18.

We examine ionizing radiation (IR) sensitivity and epistasis relationships of several Saccharomyces mutants affecting post-translational modifications of histones H2B and H3. Mutants bre1Delta, lge1Delta, and rtf1Delta, defective in histone H2B lysine 123 ubiquitination, show IR sensitivity equivalent to that of the dot1Delta mutant that we reported on earlier, consistent with published findings that Dot1p requires H2B K123 ubiquitination to fully methylate histone H3 K79. This implicates progressive K79 methylation rather than mono-methylation in IR resistance. The set2Delta mutant, defective in H3 K36 methylation, shows mild IR sensitivity whereas mutants that abolish H3 K4 methylation resemble wild type. The dot1Delta, bre1Delta, and lge1Delta mutants show epistasis for IR sensitivity. The paf1Delta mutant, also reportedly defective in H2B K123 ubiquitination, confers no sensitivity. The rad6Delta, rad51null, rad50Delta, and rad9Delta mutations are epistatic to bre1Delta and dot1Delta, but rad18Delta and rad5Delta show additivity with bre1Delta, dot1Delta, and each other. The bre1Delta rad18Delta double mutant resembles rad6Delta in sensitivity; thus the role of Rad6p in ubiquitinating H2B accounts for its extra sensitivity compared to rad18Delta. We conclude that IR resistance conferred by BRE1 and DOT1 is mediated through homologous recombinational repair, not postreplication repair, and confirm findings of a G1 checkpoint role for the RAD6/BRE1/DOT1 pathway.

X-ray survival characteristics and genetic analysis for nine Saccharomyces deletion mutants that show altered radiation sensitivity.

The availability of a genome-wide set of Saccharomyces deletion mutants provides a chance to identify all the yeast genes involved in DNA repair. Using X rays, we are screening these mutants to identify additional genes that cause increased sensitivity to the lethal effects of ionizing radiation. For each mutant identified as sensitive, we are confirming that the sensitivity phenotype cosegregates with the deletion allele and are obtaining multipoint survival-vs.-dose assays in at least one homozygous diploid and two haploid strains. We present data for deletion mutants involving the genes DOT1, MDM20, NAT3, SPT7, SPT20, GCN5, HFI1, DCC1, and VID21/EAF1 and discuss their potential roles in repair. Eight of these genes cause a clear radiation-sensitive phenotype when deleted, but the ninth, GCN5, results in at most a borderline phenotype. None of the deletions confer substantial sensitivity to ultraviolet radiation, although one or two may confer marginal sensitivity. The DOT1 gene is of interest because its only known function is to methylate one lysine residue in the core of the histone H3 protein. We find that histone H3 mutants (supplied by K. Struhl) in which this residue is replaced by other amino acids are also X-ray sensitive, which confirms that methylation of the lysine-79 residue is required for effective repair of radiation damage.

Use of a genome-wide approach to identify new genes that control resistance of Saccharomyces cerevisiae to ionizing radiation.

We have used the recently completed set of all homozygous diploid deletion mutants in budding yeast, S. cerevisiae, to screen for new mutants conferring sensitivity to ionizing radiation. In each strain a different open reading frame (ORF) has been replaced with a cassette containing unique 20-mer sequences that allow the relative abundance of each strain in a pool to be determined by hybridization to a high-density oligonucleotide array. Putative radiation-sensitive mutants were identified as having a reduced abundance in the pool of 4,627 individual deletion strains after irradiation. Of the top 33 strains most sensitive to radiation in this assay, 14 contained genes known to be involved in DNA repair. Eight of the remaining deletion mutants were studied. Only one, which deleted for the ORF YDR014W (which we name RAD61), conferred reproducible radiation sensitivity in both the haploid and diploid deletions and had no problem with spore viability when the haploid was backcrossed to wild-type. The rest showed only marginal sensitivity as haploids, and many had problems with spore viability when backcrossed, suggesting the presence of gross aneuploidy or polyploidy in strains initially presumed haploid. Our results emphasize that secondary mutations or deviations from euploidy can be a problem in screening this resource for sensitivity to ionizing radiation.

New genome-wide methods bring more power to yeast as a model organism.

Recent developments in genetic research using Saccharomyces cerevisiae have provided rapid new methods for identifying the genes that control cellular responses to treatment with a wide variety of agents, including chemicals and radiation. This significantly increases the power of yeast as a model system for studying human cellular responses to these agents, and for identifying human genes that are important in DNA repair and cancer.