Inflammation-Induced Cell Proliferation Potentiates DNA Damage-Induced Mutations In Vivo

Mutations are a critical driver of cancer initiation. While extensive studies have focused on exposure-induced mutations, few studies have explored the importance of tissue physiology as a modulator of mutation susceptibility in vivo. Of particular interest is inflammation, a known cancer risk factor relevant to chronic inflammatory diseases and pathogen-induced inflammation. Here, we used the fluorescent yellow direct repeat (FYDR) mice that harbor a reporter to detect misalignments during homologous recombination (HR), an important class of mutations. FYDR mice were exposed to cerulein, a potent inducer of pancreatic inflammation. We show that inflammation induces DSBs (γH2AX foci) and that several days later there is an increase in cell proliferation. While isolated bouts of inflammation did not induce HR, overlap between inflammation-induced DNA damage and inflammation-induced cell proliferation induced HR significantly. To study exogenously-induced DNA damage, animals were exposed to methylnitrosourea, a model alkylating agent that creates DNA lesions relevant to both environmental exposures and cancer chemotherapy. We found that exposure to alkylation damage induces HR, and importantly, that inflammation-induced cell proliferation and alkylation induce HR in a synergistic fashion. Taken together, these results show that, during an acute bout of inflammation, there is a kinetic barrier separating DNA damage from cell proliferation that protects against mutations, and that inflammation-induced cell proliferation greatly potentiates exposure-induced mutations. These studies demonstrate a fundamental mechanism by which inflammation can act synergistically with DNA damage to induce mutations that drive cancer and cancer recurrence.     

Poly(ADP-RIBOSE) polymerase-1 (Parp-1) antagonizes topoisomerase I-dependent recombination stimulation by P53

PARP-1 interacts with and poly(ADP-ribosyl)ates p53 and topoisomerase I, which both participate in DNA recombination. Previously, we showed that PARP-1 downregulates homology-directed double-strand break (DSB) repair. We also discovered that, despite the well-established role of p53 as a global suppressor of error-prone recombination, p53 enhances homologous recombination (HR) at the RARα breakpoint cluster region (bcr) comprising topoisomerase I recognition sites. Using an SV40-based assay and isogenic cell lines differing in the p53 and PARP-1 status we demonstrate that PARP-1 counteracts HR enhancement by p53, although DNA replication was largely unaffected. When the same DNA element was integrated in an episomal recombination plasmid, both p53 and PARP-1 exerted anti-recombinogenic rather than stimulatory activities. Strikingly, with DNA substrates integrated into cellular chromosomes, enhancement of HR by p53 and antagonistic PARP-1 action was seen, very similar to the HR of viral minichromosomes. siRNA-mediated knockdown revealed the essential role of topoisomerase I in this regulatory mechanism. However, after I-SceI-meganuclease-mediated cleavage of the chromosomally integrated substrate, no topoisomerase I-dependent effects by p53 and PARP-1 were observed. Our data further indicate that PARP-1, probably through topoisomerase I interactions rather than poly(ADP-ribosyl)ation, prevents p53 from stimulating spontaneous HR on chromosomes via topoisomerase I activity.     

Tissue-specific differences in the accumulation of sequence rearrangements with age

Mitotic homologous recombination (HR) is a critical pathway for the accurate repair of DNA double strand breaks (DSBs) and broken replication forks. While generally error-free, HR can occur between misaligned sequences, resulting in deleterious sequence rearrangements that can contribute to cancer and aging. To learn more about the extent to which HR occurs in different tissues during the aging process, we used Fluorescent Yellow Direct Repeat (FYDR) mice in which an HR event in a transgene yields a fluorescent phenotype. Here, we show tissue-specific differences in the accumulation of recombinant cells with age. Unlike pancreas, which shows a dramatic 23-fold increase in recombinant cell frequency with age, skin shows no increase in vivo. In vitro studies indicate that juvenile and aged primary fibroblasts are similarly able to undergo HR in response to endogenous and exogenous DNA damage. Therefore, the lack of recombinant cell accumulation in the skin is most likely not due to an inability to undergo de novo HR events. We propose that tissue-specific differences in the accumulation of recombinant cells with age result from differences in the ability of recombinant cells to persist and clonally expand within tissues.     

KAP1 Deacetylation by SIRT1 Promotes Non-Homologous End-Joining Repair

Homologous recombination and non-homologous end joining are two major DNA double-strand-break repair pathways. While HR-mediated repair requires a homologous sequence as the guiding template to restore the damage site precisely, NHEJ-mediated repair ligates the DNA lesion directly and increases the risk of losing nucleotides. Therefore, how a cell regulates the balance between HR and NHEJ has become an important issue for maintaining genomic integrity over time. Here we report that SIRT1-dependent KAP1 deacetylation positively regulates NHEJ. We show that up-regulation of KAP1 attenuates HR efficiency while promoting NHEJ repair. Moreover, SIRT1-mediated KAP1 deacetylation further enhances the effect of NHEJ by stabilizing its interaction with 53BP1, which leads to increased 53BP1 focus formation in response to DNA damage. Taken together, our study suggests a SIRT1-KAP1 regulatory mechanism for HR-NHEJ repair pathway choice.     

Dissecting the role of p53 phosphorylation in homologous recombination provides new clues for gain-of-function mutants

Regulation of homologous recombination (HR) represents the best-characterized DNA repair function of p53. The role of p53 phosphorylation in DNA repair is largely unknown. Here, we show that wild-type p53 repressed repair of DNA double-strand breaks (DSBs) by HR in a manner partially requiring the ATM/ATR phosphorylation site, serine 15. Cdk-mediated phosphorylation of serine 315 was dispensable for this anti-recombinogenic effect. However, without targeted cleavage of the HR substrate, serine 315 phosphorylation was necessary for the activation of topoisomerase I-dependent HR by p53. Moreover, overexpression of cyclin A1, which mimics the situation in tumors, inappropriately stimulated DSB-induced HR in the presence of oncogenic p53 mutants (not Wtp53). This effect required cyclin A1/cdk-mediated phosphorylation for stable complex formation with topoisomerase I. We conclude that p53 mutants have lost the balance between activation and repression of HR, which results in a net increase of potentially mutagenic DNA rearrangements. Our data provide new insight into the mechanism underlying gain-of-function of mutant p53 in genomic instability.     

BRCA1 functions independently of homologous recombination in DNA interstrand crosslink repair

Brca1 is required for DNA repair by homologous recombination (HR) and normal embryonic development. Here we report that deletion of the DNA damage response factor 53BP1 overcomes embryonic lethality in Brca1-nullizygous mice and rescues HR deficiency, as measured by hypersensitivity to polyADP-ribose polymerase (PARP) inhibition. However, Brca1,53BP1 double-deficient cells are hypersensitive to DNA interstrand crosslinks (ICLs), indicating that BRCA1 has an additional role in DNA crosslink repair that is distinct from HR. Disruption of the nonhomologous end-joining (NHEJ) factor, Ku, promotes DNA repair in Brca1-deficient cells; however deletion of either Ku or 53BP1 exacerbates genomic instability in cells lacking FANCD2, a mediator of the Fanconi anemia pathway for ICL repair. BRCA1 therefore has two separate roles in ICL repair that can be modulated by manipulating NHEJ, whereas FANCD2 provides a key activity that cannot be bypassed by ablation of 53BP1 or Ku.     

IKZF1 selectively enhances homologous recombination repair by interacting with CtIP and USP7 in multiple myeloma

Rationale: In multiple myeloma (MM), the activities of non-homologous end joining (NHEJ) and homologous recombination repair (HR) are increased compared with healthy controls. Whether and how IKZF1 as an enhancer of MM participates in the DNA repair pathway of tumor cells remains elusive.Methods: We used an endonuclease AsiSI-based system and quantitative chromatin immunoprecipitation assay (qChIP) analysis to test whether IKZF1 is involved in DNA repair. Immunopurification and mass spectrometric (MS) analysis were performed in MM1.S cells to elucidate the molecular mechanism that IKZF1 promotes DNA damage repair. The combination effect of lenalidomide or USP7 inhibitor with PARP inhibitor on cell proliferation was evaluated using MM cells in vitro and in vivo.Results: We demonstrate that IKZF1 specifically promotes homologous recombination DNA damage repair in MM cells, which is regulated by its interaction with CtIP and USP7. In this process, USP7 could regulate the stability of IKZF1 through its deubiquitinating activity. The N-terminal zinc finger domains of IKZF1 and the ubiquitin-like domain of USP7 are necessary for their interaction. Furthermore, targeted inhibition IKZF1 or USP7 could sensitize MM cells to PARP inhibitor treatment in vitro and in vivo.Conclusions: Our findings identify USP7 as a deubiquitinating enzyme for IKZF1 and uncover a new function of IKZF1 in DNA damage repair. In translational perspective, the combination inhibition of IKZF1 or USP7 with PARP inhibitor deserves further evaluation in clinical trials for the treatment of MM.     

Involvement of p54(nrb), a PSF partner protein,in DNA double-strand break repair and radioresistance

Mammalian cells repair DNA double-strand breaks (DSBs) via efficient pathways of direct, nonhomologous DNA end joining (NHEJ) and homologous recombination (HR). Prior work has identified a complex of two polypeptides, PSF and p54(nrb), as a stimulatory factor in a reconstituted in vitro NHEJ system. PSF also stimulates early steps of HR in vitro. PSF and p54(nrb) are RNA recognition motif-containing proteins with well-established functions in RNA processing and transport, and their apparent involvement in DSB repair was unexpected. Here we investigate the requirement for p54(nrb) in DSB repair in vivo. Cells treated with siRNA to attenuate p54(nrb) expression exhibited a delay in DSB repair in a γ-H2AX focus assay. Stable knockdown cell lines derived by p54(nrb) miRNA transfection showed a significant increase in ionizing radiation-induced chromosomal aberrations. They also showed increased radiosensitivity in a clonogenic survival assay. Together, results indicate that p54(nrb) contributes to rapid and accurate repair of DSBs in vivo in human cells and that the PSF·p54(nrb) complex may thus be a potential target for radiosensitizer development.     

p53 is linked directly to homologous recombination processes via RAD51/RecA protein interaction.

The tumour suppressor p53 prevents tumour formation after DNA damage by halting cell cycle progression to allow DNA repair or by inducing apoptotic cell death. Loss of wild-type p53 function renders cells resistant to DNA damage-induced cell cycle arrest and ultimately leads to genomic instabilities including gene amplifications, translocations and aneuploidy. Some of these chromosomal lesions are based on mechanisms that involve recombinatorial events. Here we report that p53 physically interacts with key factors of homologous recombination: the human RAD51 protein and its prokaryotic homologue RecA. In vitro, wild-type p53 inhibits defined biochemical activities of RecA protein, such as three-way DNA strand exchange and single strand DNA-dependent ATPase activity. In vivo, temperature-sensitive p53 forms complexes with RAD51 only in wild-type but not in mutant conformation. These observations suggest that functional wild-type p53 may select directly the appropriate pathway for DNA repair and control the extent and timing of the production of genetic variation via homologous recombination. Gene amplification an other types of chromosome rearrangements involved in tumour progression might occur not only as result of inappropriate cell proliferation but as a direct consequence of a defect in p53-mediated control of homologous recombination processes due to mutations in the p53 gene.     

BRIT1/MCPH1 Is Essential for Mitotic and Meiotic Recombination DNA Repair and Maintaining Genomic Stability in Mice

BRIT1 protein (also known as MCPH1) contains 3 BRCT domains which are conserved in BRCA1, BRCA2, and other important molecules involved in DNA damage signaling, DNA repair, and tumor suppression. BRIT1 mutations or aberrant expression are found in primary microcephaly patients as well as in cancer patients. Recent in vitro studies suggest that BRIT1/MCPH1 functions as a novel key regulator in the DNA damage response pathways. To investigate its physiological role and dissect the underlying mechanisms, we generated BRIT1 ^−/− mice and identified its essential roles in mitotic and meiotic recombination DNA repair and in maintaining genomic stability. Both BRIT1 ^−/− mice and mouse embryonic fibroblasts (MEFs) were hypersensitive to γ-irradiation. BRIT1 ^−/− MEFs and T lymphocytes exhibited severe chromatid breaks and reduced RAD51 foci formation after irradiation. Notably, BRIT1 ^−/− mice were infertile and meiotic homologous recombination was impaired. BRIT1-deficient spermatocytes exhibited a failure of chromosomal synapsis, and meiosis was arrested at late zygotene of prophase I accompanied by apoptosis. In mutant spermatocytes, DNA double-strand breaks (DSBs) were formed, but localization of RAD51 or BRCA2 to meiotic chromosomes was severely impaired. In addition, we found that BRIT1 could bind to RAD51/BRCA2 complexes and that, in the absence of BRIT1, recruitment of RAD51 and BRCA2 to chromatin was reduced while their protein levels were not altered, indicating that BRIT1 is involved in mediating recruitment of RAD51/BRCA2 to the damage site. Collectively, our BRIT1-null mouse model demonstrates that BRIT1 is essential for maintaining genomic stability in vivo to protect the hosts from both programmed and irradiation-induced DNA damages, and its depletion causes a failure in both mitotic and meiotic recombination DNA repair via impairing RAD51/BRCA2''s function and as a result leads to infertility and genomic instability in mice.     

The consequences of Rad51 overexpression for normal and tumor cells

The Rad51 recombinase is an essential factor for homologous recombination and the repair of DNA double strand breaks, binding transiently to both single stranded and double stranded DNA during the recombination reaction. The use of a homologous recombination mechanism to repair DNA damage is controlled at several levels, including the binding of Rad51 to single stranded DNA to form the Rad51 nucleofilament, which is controlled through the action of DNA helicases that can counteract nucleofilament formation. Overexpression of Rad51 in different organisms and cell types has a wide assortment of consequences, ranging from increased homologous recombination and increased resistance to DNA damaging agents to disruption of the cell cycle and apoptotic cell death. Rad51 expression is increased in p53-negative cells, and since p53 is often mutated in tumor cells, there is a tendency for Rad51 to be overexpressed in tumor cells, leading to increased resistance to DNA damage and drugs used in chemotherapies. As cells with increased Rad51 levels are more resistant to DNA damage, there is a selection for tumor cells to have higher Rad51 levels. While increased Rad51 can provide drug resistance, it also leads to increased genomic instability and may contribute to carcinogenesis.     

Regulation of rDNA stability by sumoylation

Repair of DNA lesions by homologous recombination relies on the copying of genetic information from an intact homologous sequence. However, many eukaryotic genomes contain repetitive sequences such as the ribosomal gene locus (rDNA), which poses a risk for illegitimate recombination. Therefore, the eukaryotic cell has evolved mechanisms to favor equal sister chromatid exchange (SCE) and suppress unequal SCE, single-strand annealing and break-induced replication. In the budding yeast Saccharomyces cerevisiae, the tight regulation of homologous recombination at the rDNA locus is dependent on the Smc5–Smc6 complex and sumoylation of Rad52, which directs DNA double-strand breaks in the rDNA to relocalize from within the nucleolus to the nucleoplasm before association with the recombination machinery. The relocalization before repair is important for maintaining rDNA stability. The focus of this review is the regulation of recombinational DNA repair at the rDNA locus by sumoylation and the Smc5–Smc6 complex in S. cerevisiae.     

The role of p53-mediated apoptosis as a crucial anti-tumor response to genomic instability: lessons from mouse models

Genomic instability is a major force driving human cancer development. A cellular safeguard against such genetic destabilization, which can ensue from defects in telomere maintenance, DNA repair, and checkpoint function, is activation of the p53 tumor suppressor protein, which commonly responds to these DNA damage signals by inducing apoptosis. If, however, p53 becomes inactivated, as is typical of many tumors and pre-cancerous lesions, then cells with compromised genome integrity pathways survive inappropriately, and the accrual of oncogenic lesions can fuel the carcinogenic process. Studies of mouse models have been instrumental in providing support for this idea. Mouse knockouts in genes important for telomere function, DNA damage checkpoint activation and DNA repair - both non-homologous end joining and homologous recombination - are prone to the development of genomic instability. As a consequence of these DNA damage signals, p53 becomes activated in cells of these mutant mice, leading to the induction of apoptosis, sometimes at the expense of organismal viability. This apoptotic response can be rescued through crosses to p53-deficient mice, but has dire consequences: mice predisposed to genomic instability and lacking p53 are susceptible to tumorigenesis. Thus p53-mediated apoptosis provides a crucial tumor suppressive mechanism to eliminate cells succumbing to genomic instability.