DNA-PK phosphorylation of RPA32 Ser4/Ser8 regulates replication stress checkpoint activation, fork restart, homologous recombination and mitotic catastrophe

Ashley, Amanda K.; Shrivastav, Meena; Nie, Jing; Amerin, Courtney; Troksa, Kyle; Glanzer, Jason G.; Liu, Shengqin; Opiyo, Stephen O.; Dimitrova, Diana D.; Le, Phuong N.; Sishc, Brock J.; Bailey, Susan M.; Oakley, Gregory G.; Nickoloff, Jac A.

doi:10.1016/j.dnarep.2014.04.008

Cited by 113 publications

(110 citation statements)

References 61 publications

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“…In response to replication stress, single-stranded DNA (ssDNA)-coating trimeric RPA protein is recruited and phosphorylated at multiple sites, with RPA32 S4/8 being found in the most hyperphosphorylated form of the protein (18). We observed an increase in phosphorylated RPA upon gemcitabine þ CHK1i treatment but not with either agent alone ( Supplementary Fig.…”

Section: Chk1 Inhibition Triggers Global Replication Stress By Deregumentioning

confidence: 85%

CHK1 Inhibition Synergizes with Gemcitabine Initially by Destabilizing the DNA Replication Apparatus

et al. 2015

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Combining cell-cycle checkpoint kinase inhibitors with the DNA-damaging chemotherapeutic agent gemcitabine offers clinical appeal, with a mechanistic rationale based chiefly on abrogation of gemcitabine-induced G 2 -M checkpoint activation. However, evidence supporting this mechanistic rationale from chemosensitization studies has not been consistent. Here we report a systematic definition of how pancreatic cancer cells harboring mutant p53 respond to this combination therapy, by combining mathematical models with large-scale quantitative biologic analyses of single cells and cell populations. Notably, we uncovered a dynamic range of mechanistic effects at different ratios of gemcitabine and CHK1 inhibitors. Remarkably, effective synergy was attained even where cells exhibited an apparently functional G 2 -M surveillance mechanism, as exemplified by a lack of both overt premature CDK1 activation and S-phase mitotic entry. Consistent with these findings, S-G 2 duration was extended in treated cells, leading to a definable set of lineage-dependent catastrophic fates. At synergistic drug concentrations, global replication stress was a distinct indicator of chemosensitization as characterized molecularly by an accumulation of S-phase cells with high levels of hyperphosphorylated RPA-loaded single-stranded DNA. In a fraction of these cells, persistent genomic damage was observed, including chromosomal fragmentation with a loss of centromeric regions that prevented proper kinetochore-microtubule attachment. Together, our results suggested a "foot-in-the-door" mechanism for drug synergy where cells were destroyed not by frank G 2 -M phase abrogation but rather by initiating a cumulative genotoxicity that deregulated DNA synthesis. Cancer Res; 75(17); 3583-95. Ó2015 AACR.

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Section: Chk1 Inhibition Triggers Global Replication Stress By Deregumentioning

confidence: 85%

CHK1 Inhibition Synergizes with Gemcitabine Initially by Destabilizing the DNA Replication Apparatus

et al. 2015

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“…Mutated Ser33A, Ser4/Ser8 and Thr21A phosphorylation sites in RPA2 lead to defective recovery from replication stress (Ashley et al, 2014;Olson et al, 2006). Decreased BCAS2 affects the phosphorylation of RPA2 Ser4/ Ser8 (Wan and Huang, 2014), which is phosphorylated after Thr21 and Ser33 phosphorylation (Anantha et al, 2007;Liu et al, 2012).…”

Section: Bcas2 Functions In Mouse Zygotes Through the Rpa Complexmentioning

confidence: 99%

Maternal BCAS2 protects genomic integrity in mouse early embryonic development

Wang

Xiang

et al. 2015

Development

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Mammalian early embryos maintain accurate genome integrity for proper development within a programmed timeline despite constant assaults on their DNA by replication, DNA demethylation and genetic defects transmitted from germ cells. However, how genome integrity is safeguarded during mammalian early embryonic development remains unclear. BCAS2 (breast carcinoma amplified sequence 2), a core component of the PRP19 complex involved in pre-mRNA splicing, plays an important role in the DNA damage response through the RPA complex, a key regulator in the maintenance of genome integrity. Currently, the physiological role of BCAS2 in mammals is unknown. We now report that BCAS2 responds to endogenous and exogenous DNA damage in mouse zygotes. Maternal depletion of BCAS2 compromises the DNA damage response in early embryos, leading to developmental arrest at the two-to four-cell stage accompanied by the accumulation of damaged DNA and micronuclei. Furthermore, BCAS2 mutants that are unable to bind RPA1 fail in DNA repair during the zygotic stage. In addition, phosphorylated RPA2 cannot localise to the DNA damage sites in mouse zygotes with disrupted maternal BCAS2. These data suggest that BCAS2 might function through the RPA complex during DNA repair in zygotes. Together, our results reveal that maternal BCAS2 maintains the genome integrity of early embryos and is essential for female mouse fertility.

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“…For example, the S4/8A mutant is defective in Chk1, Mre11 and TopBP1 phosphorylation in response to etoposide [98]. Recently, it was shown that cells expressing the S4/8A mutant restart replication prematurely during the recovery from etoposide-induced DNA damage, indicating a defect in sustaining the proper checkpoint response [107]. Cells lacking DNA-PKcs or expressing a kinase-inactive DNA-PKcs mutant also had decreased Chk1 phosphorylation, thereby implicating DNA-PK in ATR activation.…”

Section: Rpa Phosphorylation and The Ddrmentioning

confidence: 99%

RPA-coated single-stranded DNA as a platform for post-translational modifications in the DNA damage response

2014

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The Replication Protein A (RPA) complex is an essential regulator of eukaryotic DNA metabolism. RPA avidly binds to single-stranded DNA (ssDNA) through multiple oligonucleotide/oligosaccharide-binding folds and coordinates the recruitment and exchange of genome maintenance factors to regulate DNA replication, recombination and repair. The RPA-ssDNA platform also constitutes a key physiological signal which activates the master ATR kinase to protect and repair stalled or collapsed replication forks during replication stress. In recent years, the RPA complex has emerged as a key target and an important regulator of post-translational modifications in response to DNA damage, which is critical for its genome guardian functions. Phosphorylation and SUMOylation of the RPA complex, and more recently RPA-regulated ubiquitination, have all been shown to control specific aspects of DNA damage signaling and repair by modulating the interactions between RPA and its partners. Here, we review our current understanding of the critical functions of the RPA-ssDNA platform in the maintenance of genome stability and its regulation through an elaborate network of covalent modifications.

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DNA-PK phosphorylation of RPA32 Ser4/Ser8 regulates replication stress checkpoint activation, fork restart, homologous recombination and mitotic catastrophe

Cited by 113 publications

References 61 publications

CHK1 Inhibition Synergizes with Gemcitabine Initially by Destabilizing the DNA Replication Apparatus

CHK1 Inhibition Synergizes with Gemcitabine Initially by Destabilizing the DNA Replication Apparatus

Maternal BCAS2 protects genomic integrity in mouse early embryonic development

RPA-coated single-stranded DNA as a platform for post-translational modifications in the DNA damage response

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