Genome Protection by the 9-1-1 Complex Subunit HUS1 Requires Clamp Formation, DNA Contacts, and ATR Signaling-independent Effector Functions.

The RAD9A-HUS1-RAD1 (9-1-1) complex is a heterotrimeric clamp that promotes checkpoint signaling and repair at DNA damage sites. In this study, we elucidated HUS1 functional residues that drive clamp assembly, DNA interactions, and downstream effector functions. First, we mapped a HUS1-RAD9A interface residue that was critical for 9-1-1 assembly and DNA loading. Next, we identified multiple positively charged residues in the inner ring of HUS1 that were crucial for genotoxin-induced 9-1-1 chromatin localization and ATR signaling. Finally, we found two hydrophobic pockets on the HUS1 outer surface that were important for cell survival after DNA damage. Interestingly, these pockets were not required for 9-1-1 chromatin localization or ATR-mediated CHK1 activation but were necessary for interactions between HUS1 and its binding partner MYH, suggesting that they serve as interaction domains for the recruitment and coordination of downstream effectors at damage sites. Together, these results indicate that, once properly loaded onto damaged DNA, the 9-1-1 complex executes multiple, separable functions that promote genome maintenance.

Conditional inactivation of the DNA damage response gene Hus1 in mouse testis reveals separable roles for components of the RAD9-RAD1-HUS1 complex in meiotic chromosome maintenance.

The RAD9-RAD1-HUS1 (9-1-1) complex is a heterotrimeric PCNA-like clamp that responds to DNA damage in somatic cells by promoting DNA repair as well as ATR-dependent DNA damage checkpoint signaling. In yeast, worms, and flies, the 9-1-1 complex is also required for meiotic checkpoint function and efficient completion of meiotic recombination; however, since Rad9, Rad1, and Hus1 are essential genes in mammals, little is known about their functions in mammalian germ cells. In this study, we assessed the meiotic functions of 9-1-1 by analyzing mice with germ cell-specific deletion of Hus1 as well as by examining the localization of RAD9 and RAD1 on meiotic chromosomes during prophase I. Hus1 loss in testicular germ cells resulted in meiotic defects, germ cell depletion, and severely compromised fertility. Hus1-deficient primary spermatocytes exhibited persistent autosomal γH2AX and RAD51 staining indicative of unrepaired meiotic DSBs, synapsis defects, an extended XY body domain often encompassing partial or whole autosomes, and an increase in structural chromosome abnormalities such as end-to-end X chromosome-autosome fusions and ruptures in the synaptonemal complex. Most of these aberrations persisted in diplotene-stage spermatocytes. Consistent with a role for the 9-1-1 complex in meiotic DSB repair, RAD9 localized to punctate, RAD51-containing foci on meiotic chromosomes in a Hus1-dependent manner. Interestingly, RAD1 had a broader distribution that only partially overlapped with RAD9, and localization of both RAD1 and the ATR activator TOPBP1 to the XY body and to unsynapsed autosomes was intact in Hus1 conditional knockouts. We conclude that mammalian HUS1 acts as a component of the canonical 9-1-1 complex during meiotic prophase I to promote DSB repair and further propose that RAD1 and TOPBP1 respond to unsynapsed chromatin through an alternative mechanism that does not require RAD9 or HUS1.

Disease severity in a mouse model of ataxia telangiectasia is modulated by the DNA damage checkpoint gene Hus1.

The human genomic instability syndrome ataxia telangiectasia (A-T), caused by mutations in the gene encoding the DNA damage checkpoint kinase ATM, is characterized by multisystem defects including neurodegeneration, immunodeficiency and increased cancer predisposition. ATM is central to a pathway that responds to double-strand DNA breaks, whereas the related kinase ATR leads a parallel signaling cascade that is activated by replication stress. To dissect the physiological relationship between the ATM and ATR pathways, we generated mice defective for both. Because complete ATR pathway inactivation causes embryonic lethality, we weakened the ATR mechanism to different degrees by impairing HUS1, a member of the 911 complex that is required for efficient ATR signaling. Notably, simultaneous ATM and HUS1 defects caused synthetic lethality. Atm/Hus1 double-mutant embryos showed widespread apoptosis and died mid-gestationally. Despite the underlying DNA damage checkpoint defects, increased DNA damage signaling was observed, as evidenced by H2AX phosphorylation and p53 accumulation. A less severe Hus1 defect together with Atm loss resulted in partial embryonic lethality, with the surviving double-mutant mice showing synergistic increases in genomic instability and specific developmental defects, including dwarfism, craniofacial abnormalities and brachymesophalangy, phenotypes that are observed in several human genomic instability disorders. In addition to identifying tissue-specific consequences of checkpoint dysfunction, these data highlight a robust, cooperative configuration for the mammalian DNA damage response network and further suggest HUS1 and related genes in the ATR pathway as candidate modifiers of disease severity in A-T patients.

Multiple 9-1-1 complexes promote homolog synapsis, DSB repair, and ATR signaling during mammalian meiosis.

DNA damage response mechanisms have meiotic roles that ensure successful gamete formation. While completion of meiotic double-strand break (DSB) repair requires the canonical RAD9A-RAD1-HUS1 (9A-1-1) complex, mammalian meiocytes also express RAD9A and HUS1 paralogs, RAD9B and HUS1B, predicted to form alternative 9-1-1 complexes. The RAD1 subunit is shared by all predicted 9-1-1 complexes and localizes to meiotic chromosomes even in the absence of HUS1 and RAD9A. Here, we report that testis-specific disruption of RAD1 in mice resulted in impaired DSB repair, germ cell depletion, and infertility. Unlike Hus1 or Rad9a disruption, Rad1 loss in meiocytes also caused severe defects in homolog synapsis, impaired phosphorylation of ATR targets such as H2AX, CHK1, and HORMAD2, and compromised meiotic sex chromosome inactivation. Together, these results establish critical roles for both canonical and alternative 9-1-1 complexes in meiotic ATR activation and successful prophase I completion.

A tale of tails: insights into the coordination of 3' end processing during homologous recombination.

Eukaryotic genomes harbor a large number of homologous repeat sequences that are capable of recombining. Their potential to disrupt genome stability highlights the need to understand how homologous recombination processes are coordinated. The Saccharomyces cerevisiae Rad1-Rad10 endonuclease performs an essential role in recombination between repeated sequences, by processing 3' single-stranded intermediates formed during single-strand annealing and gene conversion events. Several recent studies have focused on factors involved in Rad1-Rad10-dependent removal of 3' nonhomologous tails during homologous recombination, including Msh2-Msh3, Slx4, and the newly identified Saw1 protein. Together, this new work provides a model for how Rad1-Rad10-dependent end processing is coordinated: Msh2-Msh3 stabilizes and prepares double-strand/single-strand junctions for Rad1-Rad10 cleavage, Saw1 recruits Rad1-Rad10 to 3' tails, and Slx4 mediates crosstalk between the DNA damage checkpoint machinery and Rad1-Rad10.

A Genetically Engineered Mouse Model of Malignant Testicular Germ Cell Tumors.

Testicular germ cell tumors (TGCTs) are among the most curable solid cancers and are typically highly responsive to conventional DNA-damaging chemotherapies, even in patients with metastatic disease. It has therefore been of great interest to understand the basis for the unique chemosensitivity of these cancers, which is linked to the DNA damage sensitivity of their cancer stem cells. TGCTs have been difficult to study in the mouse, however, since most of the existing mouse models develop benign teratomas that are unlike the malignant TGCTs that afflict most testicular cancer patients. We describe here methods for generating a TGCT mouse model that closely resembles the malignant, metastatic disease observed in men with testicular cancer, and additionally include methods for analyzing the cancer stems cells and responses to chemotherapeutics in these murine TGCTs.

Mutants defective in Rad1-Rad10-Slx4 exhibit a unique pattern of viability during mating-type switching in Saccharomyces cerevisiae.

Efficient repair of DNA double-strand breaks (DSBs) requires the coordination of checkpoint signaling and enzymatic repair functions. To study these processes during gene conversion at a single chromosomal break, we monitored mating-type switching in Saccharomyces cerevisiae strains defective in the Rad1-Rad10-Slx4 complex. Rad1-Rad10 is a structure-specific endonuclease that removes 3' nonhomologous single-stranded ends that are generated during many recombination events. Slx4 is a known target of the DNA damage response that forms a complex with Rad1-Rad10 and is critical for 3'-end processing during repair of DSBs by single-strand annealing. We found that mutants lacking an intact Rad1-Rad10-Slx4 complex displayed RAD9- and MAD2-dependent cell cycle delays and decreased viability during mating-type switching. In particular, these mutants exhibited a unique pattern of dead and switched daughter cells arising from the same DSB-containing cell. Furthermore, we observed that mutations in post-replicative lesion bypass factors (mms2Delta, mph1Delta) resulted in decreased viability during mating-type switching and conferred shorter cell cycle delays in rad1Delta mutants. We conclude that Rad1-Rad10-Slx4 promotes efficient repair during gene conversion events involving a single 3' nonhomologous tail and propose that the rad1Delta and slx4Delta mutant phenotypes result from inefficient repair of a lesion at the MAT locus that is bypassed by replication-mediated repair.

Chemotherapy-Induced Depletion of OCT4-Positive Cancer Stem Cells in a Mouse Model of Malignant Testicular Cancer.

Testicular germ cell tumors (TGCTs) are among the most responsive solid cancers to conventional chemotherapy. To elucidate the underlying mechanisms, we developed a mouse TGCT model featuring germ cell-specific Kras activation and Pten inactivation. The resulting mice developed malignant, metastatic TGCTs composed of teratoma and embryonal carcinoma, the latter of which exhibited stem cell characteristics, including expression of the pluripotency factor OCT4. Consistent with epidemiological data linking human testicular cancer risk to in utero exposures, embryonic germ cells were susceptible to malignant transformation, whereas adult germ cells underwent apoptosis in response to the same oncogenic events. Treatment of tumor-bearing mice with genotoxic chemotherapy not only prolonged survival and reduced tumor size but also selectively eliminated the OCT4-positive cancer stem cells. We conclude that the chemosensitivity of TGCTs derives from the sensitivity of their cancer stem cells to DNA-damaging chemotherapy.

A Delicate Balance Between Repair and Replication Factors Regulates Recombination Between Divergent DNA Sequences in Saccharomyces cerevisiae.

Single-strand annealing (SSA) is an important homologous recombination mechanism that repairs DNA double strand breaks (DSBs) occurring between closely spaced repeat sequences. During SSA, the DSB is acted upon by exonucleases to reveal complementary sequences that anneal and are then repaired through tail clipping, DNA synthesis, and ligation steps. In baker's yeast, the Msh DNA mismatch recognition complex and the Sgs1 helicase act to suppress SSA between divergent sequences by binding to mismatches present in heteroduplex DNA intermediates and triggering a DNA unwinding mechanism known as heteroduplex rejection. Using baker's yeast as a model, we have identified new factors and regulatory steps in heteroduplex rejection during SSA. First we showed that Top3-Rmi1, a topoisomerase complex that interacts with Sgs1, is required for heteroduplex rejection. Second, we found that the replication processivity clamp proliferating cell nuclear antigen (PCNA) is dispensable for heteroduplex rejection, but is important for repairing mismatches formed during SSA. Third, we showed that modest overexpression of Msh6 results in a significant increase in heteroduplex rejection; this increase is due to a compromise in Msh2-Msh3 function required for the clipping of 3' tails. Thus 3' tail clipping during SSA is a critical regulatory step in the repair vs. rejection decision; rejection is favored before the 3' tails are clipped. Unexpectedly, Msh6 overexpression, through interactions with PCNA, disrupted heteroduplex rejection between divergent sequences in another recombination substrate. These observations illustrate the delicate balance that exists between repair and replication factors to optimize genome stability.

Clamping down on mammalian meiosis.

The RAD9A-RAD1-HUS1 (9-1-1) complex is a PCNA-like heterotrimeric clamp that binds damaged DNA to promote cell cycle checkpoint signaling and DNA repair. While various 9-1-1 functions in mammalian somatic cells have been established, mounting evidence from lower eukaryotes predicts critical roles in meiotic germ cells as well. This was investigated in 2 recent studies in which the 9-1-1 complex was disrupted specifically in the mouse male germline through conditional deletion of Rad9a or Hus1. Loss of these clamp subunits led to severely impaired fertility and meiotic defects, including faulty DNA double-strand break repair. While 9-1-1 is critical for ATR kinase activation in somatic cells, these studies did not reveal major defects in ATR checkpoint pathway signaling in meiotic cells. Intriguingly, this new work identified separable roles for 9-1-1 subunits, namely RAD9A- and HUS1-independent roles for RAD1. Based on these studies and the high-level expression of the paralogous proteins RAD9B and HUS1B in testis, we propose a model in which multiple alternative 9-1-1 clamps function during mammalian meiosis to ensure genome maintenance in the germline.

The DNA damage checkpoint allows recombination between divergent DNA sequences in budding yeast.

In the early steps of homologous recombination, single-stranded DNA (ssDNA) from a broken chromosome invades homologous sequence located in a sister or homolog donor. In genomes that contain numerous repetitive DNA elements or gene paralogs, recombination can potentially occur between non-allelic/divergent (homeologous) sequences that share sequence identity. Such recombination events can lead to lethal chromosomal deletions or rearrangements. However, homeologous recombination events can be suppressed through rejection mechanisms that involve recognition of DNA mismatches in heteroduplex DNA by mismatch repair factors, followed by active unwinding of the heteroduplex DNA by helicases. Because factors required for heteroduplex rejection are hypothesized to be targets and/or effectors of the DNA damage response (DDR), a cell cycle control mechanism that ensures timely and efficient repair, we tested whether the DDR, and more specifically, the RAD9 gene, had a role in regulating rejection. We performed these studies using a DNA repair assay that measures repair by single-strand annealing (SSA) of a double-strand break (DSB) using homeologous DNA templates. We found that repair of homeologous DNA sequences, but not identical sequences, induced a RAD9-dependent cell cycle delay in the G2 stage of the cell cycle. Repair through a divergent DNA template occurred more frequently in RAD9 compared to rad9Δ strains. However, repair in rad9Δ mutants could be restored to wild-type levels if a G2 delay was induced by nocodazole. These results suggest that cell cycle arrest induced by the Rad9-dependent DDR allows repair between divergent DNA sequences despite the potential for creating deleterious genome rearrangements, and illustrates the importance of additional cellular mechanisms that act to suppress recombination between divergent DNA sequences.