Replication stress triggers microsatellite destabilization and hypermutation leading to clonal expansion in vitro.

Mismatch repair (MMR)-deficient cancers are characterized by microsatellite instability (MSI) and hypermutation. However, it remains unclear how MSI and hypermutation arise and contribute to cancer development. Here, we show that MSI and hypermutation are triggered by replication stress in an MMR-deficient background, enabling clonal expansion of cells harboring ARF/p53-module mutations and cells that are resistant to the anti-cancer drug camptothecin. While replication stress-associated DNA double-strand breaks (DSBs) caused chromosomal instability (CIN) in an MMR-proficient background, they induced MSI with concomitant suppression of CIN via a PARP-mediated repair pathway in an MMR-deficient background. This was associated with the induction of mutations, including cancer-driver mutations in the ARF/p53 module, via chromosomal deletions and base substitutions. Immortalization of MMR-deficient mouse embryonic fibroblasts (MEFs) in association with ARF/p53-module mutations was ~60-fold more efficient than that of wild-type MEFs. Thus, replication stress-triggered MSI and hypermutation efficiently lead to clonal expansion of cells with abrogated defense systems.

Mismatch repair dependence of replication stress-associated DSB recognition and repair.

Most cancers develop with one of two types of genomic instability, namely, chromosomal instability (CIN) or microsatellite instability (MSI). Both are induced by replication stress-associated DNA double-strand breaks (DSBs). The type of genomic instability that arises is dependent on the choice of DNA repair pathway. Specifically, MSI is induced via a PolQ-dependent repair pathway called microhomology-mediated end joining (MMEJ) in a mismatch repair (MMR)-deficient background. However, it is unclear how the MMR status determines the choice of DSB repair pathway. Here, we show that replication stress-associated DSBs initially targeted by the homologous recombination (HR) system were subsequently hijacked by PolQ-dependent MMEJ in MMR-deficient cells, but persisted as HR intermediates in MMR-proficient cells. PolQ interacting with MMR factors was effectively loaded onto damaged chromatin in an MMR-deficient background, in which merged MRE11/γH2AX foci also effectively formed. Thus, the choice of DNA repair pathway according to the MMR status determines whether CIN or MSI is induced.

Onset of deaminase APOBEC3B induction in response to DNA double-strand breaks.

Deamination of 5-methyl cytosine is a major cause of cancer-driver mutations in inflammation-associated cancers. The deaminase APOBEC3B is expressed in these cancers and causes mutations under replication stress; however, the mechanisms by which APOBEC3B mediates deamination and its association with genomic disorders are still unclear. Here, we show that APOBEC3B is stabilized to induce deamination reaction in response to DNA double-strand breaks (DSBs), resulting in the formation of long-lasting DSBs. Uracil, the major deamination product, is subsequently targeted by base excision repair (BER) through uracil-DNA glycosylase 2 (UNG2); hence late-onset DSBs arise as by-products of BER. The frequency of these delayed DSBs was increased by treatment of cells with a PARP inhibitor, and was suppressed following knock-down of UNG2. The late-onset DSBs were induced in an ATR-dependent manner. Those secondary DSBs were persistent, unlike DSBs directly caused by γ-ray irradiation. Overall, these results suggest that the deaminase APOBEC3B is induced in response to DSBs, leading to long-lasting DSB formation in addition to mutagenic 5me-C>T transition induction.

Gamma-irradiated quiescent cells repair directly induced double-strand breaks but accumulate persistent double-strand breaks during subsequent DNA replication.

H2AX is expressed at very low levels in quiescent normal cells in vivo and in vitro. Such cells repair DNA double-strand breaks (DSBs) induced by γ-irradiation through a transient stabilization of H2AX. However, the resultant cells accumulate small numbers of irreparable (or persistent) DSBs via an unknown mechanism. We found that quiescent cells that had repaired DSBs directly induced by γ-rays were prone to accumulate DSBs during the subsequent DNA replication. Unlike directly induced DSBs, secondary DSBs were not efficiently repaired, although Rad51 and 53BP1 were recruited to these sites. H2AX was dramatically stabilized in response to DSBs directly caused by γ-rays, enabling γH2AX foci formation and DSB repair, whereas H2AX was barely stabilized in response to secondary DSBs, in which γH2AX foci were small and DSBs were not efficiently repaired. Our results show a pathway that leads to the persistent DSB formation after γ-irradiation.

ATM and SIRT6/SNF2H Mediate Transient H2AX Stabilization When DSBs Form by Blocking HUWE1 to Allow Efficient γH2AX Foci Formation.

In response to DNA double-strand breaks (DSBs), H2AX is rapidly phosphorylated at Ser139 to promote DSB repair. Here we show that H2AX is rapidly stabilized in response to DSBs to efficiently generate γH2AX foci. This mechanism operated even in quiescent cells that barely expressed H2AX. H2AX stabilization resulted from the inhibition of proteasome-mediated degradation. Synthesized H2AX ordinarily underwent degradation through poly-ubiquitination mediated by the E3 ligase HUWE1; however, H2AX ubiquitination was transiently halted upon DSB formation. Such rapid H2AX stabilization by DSBs was associated with chromatin incorporation of H2AX and halting of its poly-ubiquitination mediated by the ATM kinase, the sirtuin protein SIRT6, and the chromatin remodeler SNF2H. H2AX Ser139, the ATM phosphorylation site, was essential for H2AX stabilization upon DSB formation. Our results reveal a pathway controlled by ATM, SIRT6, and SNF2H to block HUWE1, which stabilizes H2AX and induces its incorporation into chromatin only when cells are damaged.