ATM suppresses c-Myc overexpression in the mammary epithelium in response to estrogen.

ATM gene mutation carriers are predisposed to estrogen-receptor-positive breast cancer (BC). ATM prevents BC oncogenesis by activating p53 in every cell; however, much remains unknown about tissue-specific oncogenesis after ATM loss. Here, we report that ATM controls the early transcriptional response to estrogens. This response depends on topoisomerase II (TOP2), which generates TOP2-DNA double-strand break (DSB) complexes and rejoins the breaks. When TOP2-mediated ligation fails, ATM facilitates DSB repair. After estrogen exposure, TOP2-dependent DSBs arise at the c-MYC enhancer in human BC cells, and their defective repair changes the activation profile of enhancers and induces the overexpression of many genes, including the c-MYC oncogene. CRISPR/Cas9 cleavage at the enhancer also causes c-MYC overexpression, indicating that this DSB causes c-MYC overexpression. Estrogen treatment induced c-Myc protein overexpression in mammary epithelial cells of ATM-deficient mice. In conclusion, ATM suppresses the c-Myc-driven proliferative effects of estrogens, possibly explaining such tissue-specific oncogenesis.

The fragility of a structurally diverse duplication block triggers recurrent genomic amplification.

The human genome contains hundreds of large, structurally diverse blocks that are insufficiently represented in the reference genome and are thus not amenable to genomic analyses. Structural diversity in the human population suggests that these blocks are unstable in the germline; however, whether or not these blocks are also unstable in the cancer genome remains elusive. Here we report that the 500 kb block called KRTAP_region_1 (KRTAP-1) on 17q12-21 recurrently demarcates the amplicon of the ERBB2 (HER2) oncogene in breast tumors. KRTAP-1 carries numerous tandemly-duplicated segments that exhibit diversity within the human population. We evaluated the fragility of the block by cytogenetically measuring the distances between the flanking regions and found that spontaneous distance outliers (i.e DNA breaks) appear more frequently at KRTAP-1 than at the representative common fragile site (CFS) FRA16D. Unlike CFSs, KRTAP-1 is not sensitive to aphidicolin. The exonuclease activity of DNA repair protein Mre11 protects KRTAP-1 from breaks, whereas CtIP does not. Breaks at KRTAP-1 lead to the palindromic duplication of the ERBB2 locus and trigger Breakage-Fusion-Bridge cycles. Our results indicate that an insufficiently investigated area of the human genome is fragile and could play a crucial role in cancer genome evolution.

Genetic evidence for the involvement of mismatch repair proteins, PMS2 and MLH3, in a late step of homologous recombination.

Homologous recombination (HR) repairs DNA double-strand breaks using intact homologous sequences as template DNA. Broken DNA and intact homologous sequences form joint molecules (JMs), including Holliday junctions (HJs), as HR intermediates. HJs are resolved to form crossover and noncrossover products. A mismatch repair factor, MLH3 endonuclease, produces the majority of crossovers during meiotic HR, but it remains elusive whether mismatch repair factors promote HR in nonmeiotic cells. We disrupted genes encoding the MLH3 and PMS2 endonucleases in the human B cell line, TK6, generating null MLH3-/- and PMS2-/- mutant cells. We also inserted point mutations into the endonuclease motif of MLH3 and PMS2 genes, generating endonuclease death MLH3DN/DN and PMS2EK/EK cells. MLH3-/- and MLH3DN/DN cells showed a very similar phenotype, a 2.5-fold decrease in the frequency of heteroallelic HR-dependent repair of restriction enzyme-induced double-strand breaks. PMS2-/- and PMS2EK/EK cells showed a phenotype very similar to that of the MLH3 mutants. These data indicate that MLH3 and PMS2 promote HR as an endonuclease. The MLH3DN/DN and PMS2EK/EK mutations had an additive effect on the heteroallelic HR. MLH3DN/DN/PMS2EK/EK cells showed normal kinetics of γ-irradiation-induced Rad51 foci but a significant delay in the resolution of Rad51 foci and a 3-fold decrease in the number of cisplatin-induced sister chromatid exchanges. The ectopic expression of the Gen1 HJ re-solvase partially reversed the defective heteroallelic HR of MLH3DN/DN/PMS2EK/EK cells. Taken together, we propose that MLH3 and PMS2 promote HR as endonucleases, most likely by processing JMs in mammalian somatic cells.

CtIP-BRCA1 complex and MRE11 maintain replication forks in the presence of chain terminating nucleoside analogs.

Chain-terminating nucleoside analogs (CTNAs), which cannot be extended by DNA polymerases, are widely used as antivirals or anti-cancer agents, and can induce cell death. Processing of blocked DNA ends, like camptothecin-induced trapped-topoisomerase I, can be mediated by TDP1, BRCA1, CtIP and MRE11. Here, we investigated whether the CtIP-BRCA1 complex and MRE11 also contribute to cellular tolerance to CTNAs, including 2',3'-dideoxycytidine (ddC), cytarabine (ara-C) and zidovudine (Azidothymidine, AZT). We show that BRCA1-/-, CtIPS332A/-/- and nuclease-dead MRE11D20A/- mutants display increased sensitivity to CTNAs, accumulate more DNA damage (chromosomal breaks, γ-H2AX and neutral comets) when treated with CTNAs and exhibit significant delays in replication fork progression during exposure to CTNAs. Moreover, BRCA1-/-, CtIPS332A/-/- and nuclease-dead MRE11D20A/- mutants failed to resume DNA replication in response to CTNAs, whereas control and CtIP+/-/- cells experienced extensive recovery of DNA replication. In summary, we provide clear evidence that MRE11 and the collaborative action of BRCA1 and CtIP play a critical role in the nuclease-dependent removal of incorporated ddC from replicating genomic DNA. We propose that BRCA1-CTIP and MRE11 prepare nascent DNA ends, blocked from synthesis by CTNAs, for further repair.

SUMOylation of PCNA by PIAS1 and PIAS4 promotes template switch in the chicken and human B cell lines.

DNA damage tolerance (DDT) releases replication blockage caused by damaged nucleotides on template strands employing two alternative pathways, error-prone translesion DNA synthesis (TLS) and error-free template switch (TS). Lys164 of proliferating cell nuclear antigen (PCNA) is SUMOylated during the physiological cell cycle. To explore the role for SUMOylation of PCNA in DDT, we characterized chicken DT40 and human TK6 B cells deficient in the PIAS1 and PIAS4 small ubiquitin-like modifier (SUMO) E3 ligases. DT40 cells have a unique advantage in the phenotypic analysis of DDT as they continuously diversify their immunoglobulin (Ig) variable genes by TLS and TS [Ig gene conversion (GC)], both relieving replication blocks at abasic sites without accompanying by DNA breakage. Remarkably, PIAS1-/-/PIAS4-/- cells displayed a multifold decrease in SUMOylation of PCNA at Lys164 and over a 90% decrease in the rate of TS. Likewise, PIAS1-/-/PIAS4-/- TK6 cells showed a shift of DDT from TS to TLS at a chemosynthetic UV lesion inserted into the genomic DNA. The PCNAK164R/K164R mutation caused a ∼90% decrease in the rate of Ig GC and no additional impact on PIAS1-/-/PIAS4-/- cells. This epistatic relationship between the PCNAK164R/K164R and the PIAS1-/-/PIAS4-/- mutations suggests that PIAS1 and PIAS4 promote TS mainly through SUMOylation of PCNA at Lys164. This idea is further supported by the data that overexpression of a PCNA-SUMO1 chimeric protein restores defects in TS in PIAS1-/-/PIAS4-/- cells. In conclusion, SUMOylation of PCNA at Lys164 promoted by PIAS1 and PIAS4 ensures the error-free release of replication blockage during physiological DNA replication in metazoan cells.

BRCA1 ensures genome integrity by eliminating estrogen-induced pathological topoisomerase II-DNA complexes.

Women having BRCA1 germ-line mutations develop cancer in breast and ovary, estrogen-regulated tissues, with high penetrance. Binding of estrogens to the estrogen receptor (ER) transiently induces DNA double-strand breaks (DSBs) by topoisomerase II (TOP2) and controls gene transcription. TOP2 resolves catenated DNA by transiently generating DSBs, TOP2-cleavage complexes (TOP2ccs), where TOP2 covalently binds to 5' ends of DSBs. TOP2 frequently fails to complete its catalysis, leading to formation of pathological TOP2ccs. We have previously shown that the endonucleolytic activity of MRE11 plays a key role in removing 5' TOP2 adducts in G1 phase. We show here that BRCA1 promotes MRE11-mediated removal of TOP2 adducts in G1 phase. We disrupted the BRCA1 gene in 53BP1-deficient ER-positive breast cancer and B cells. The loss of BRCA1 caused marked increases of pathological TOP2ccs in G1 phase following exposure to etoposide, which generates pathological TOP2ccs. We conclude that BRCA1 promotes the removal of TOP2 adducts from DSB ends for subsequent nonhomologous end joining. BRCA1-deficient cells showed a decrease in etoposide-induced MRE11 foci in G1 phase, suggesting that BRCA1 repairs pathological TOP2ccs by promoting the recruitment of MRE11 to TOP2cc sites. BRCA1 depletion also leads to the increase of unrepaired DSBs upon estrogen treatment both in vitro in G1-arrested breast cancer cells and in vivo in epithelial cells of mouse mammary glands. BRCA1 thus plays a critical role in removing pathological TOP2ccs induced by estrogens as well as etoposide. We propose that BRCA1 suppresses tumorigenesis by removing estrogen-induced pathological TOP2ccs throughout the cell cycle.

Smarcal1 promotes double-strand-break repair by nonhomologous end-joining.

Smarcal1 is a SWI/SNF-family protein with an ATPase domain involved in DNA-annealing activities and a binding site for the RPA single-strand-DNA-binding protein. Although the role played by Smarcal1 in the maintenance of replication forks has been established, it remains unknown whether Smarcal1 contributes to genomic DNA maintenance outside of the S phase. We disrupted the SMARCAL1 gene in both the chicken DT40 and the human TK6 B cell lines. The resulting SMARCAL1(-/-) clones exhibited sensitivity to chemotherapeutic topoisomerase 2 inhibitors, just as nonhomologous end-joining (NHEJ) null-deficient cells do. SMARCAL1(-/-) cells also exhibited an increase in radiosensitivity in the G1 phase. Moreover, the loss of Smarcal1 in NHEJ null-deficient cells does not further increase their radiosensitivity. These results demonstrate that Smarcal1 is required for efficient NHEJ-mediated DSB repair. Both inactivation of the ATPase domain and deletion of the RPA-binding site cause the same phenotype as does null-mutation of Smarcal1, suggesting that Smarcal1 enhances NHEJ, presumably by interacting with RPA at unwound single-strand sequences and then facilitating annealing at DSB ends. SMARCAL1(-/-)cells showed a poor accumulation of Ku70/DNA-PKcs and XRCC4 at DNA-damage sites. We propose that Smarcal1 maintains the duplex status of DSBs to ensure proper recruitment of NHEJ factors to DSB sites.