Coupling of Human DNA Excision Repair and the DNA Damage Checkpoint in a Defined in Vitro System

Background:In quiescent human cells, UV induces histone H2AX phosphorylation by ATR in a nucleotide excision repair (NER)-dependent manner. Results: UV also activates ATM in response to NER-mediated DNA double-strand break (DSB). Conclusion:The NER reaction in quiescent cells potentially generates multiple types of secondary DNA damage. Significance: This work highlights the importance of our understanding of the DNA damage response in quiescent cells.

Section: Discussionmentioning

confidence: 99%

Nucleotide Excision Repair-dependent DNA Double-strand Break Formation and ATM Signaling Activation in Mammalian Quiescent Cells

Wakasugi

Sasaki

Matsumoto

et al. 2014

“…Cycling cells were typically plated at 30% confluence and treated the following day. Non-cycling, quiescent cells were plated at 40 -60% confluence, grown for 2 days in normal medium until the cells reached confluence, and then maintained for 2-3 days in DMEM containing 0.5% FBS and penicillin/streptomycin before being subjected to experimental treatments as previously described (32,36). To expose cells to the indicated fluences of UV radiation, media was removed and set aside before placing the cells under a GE germicidal lamp that emits primarily 254-nm UV light (UV-C) that was connected to a digital timer.…”

Section: Methodsmentioning

confidence: 99%

“…Non-cycling Cells-Previous studies comparing UV-induced DNA damage signaling in cycling and noncycling cells have been rather limited (29,32). Thus, to examine this issue in greater detail and specifically the contribution of the ATR kinase, we monitored the phosphorylation of several key DNA damage response signaling proteins following UV irradiation of rapidly growing, subconfluent HaCaT keratinocytes (cycling cells) and cells grown to confluence and maintained in low serum (non-cycling cells).…”

Section: Uv-induced Dna Damage Signaling Exhibit Different Profiles Imentioning

confidence: 99%

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ATR Kinase Inhibition Protects Non-cycling Cells from the Lethal Effects of DNA Damage and Transcription Stress

Kemp

Sancar

2016

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ATR (ataxia telangiectasia and Rad-3-related) is a protein kinase that maintains genome stability and halts cell cycle phase transitions in response to DNA lesions that block DNA polymerase movement. These DNA replication-associated features of ATR function have led to the emergence of ATR kinase inhibitors as potential adjuvants for DNA-damaging cancer chemotherapeutics. However, whether ATR affects the genotoxic stress response in non-replicating, non-cycling cells is currently unknown. We therefore used chemical inhibition of ATR kinase activity to examine the role of ATR in quiescent human cells. Although ATR inhibition had no obvious effects on the viability of non-cycling cells, inhibition of ATR partially protected nonreplicating cells from the lethal effects of UV and UV mimetics. Analyses of various DNA damage response signaling pathways demonstrated that ATR inhibition reduced the activation of apoptotic signaling by these agents in non-cycling cells. The pro-apoptosis/cell death function of ATR is likely due to transcription stress because the lethal effects of compounds that block RNA polymerase movement were reduced in the presence of an ATR inhibitor. These results therefore suggest that whereas DNA polymerase stalling at DNA lesions activates ATR to protect cell viability and prevent apoptosis, the stalling of RNA polymerases instead activates ATR to induce an apoptotic form of cell death in non-cycling cells. These results have important implications regarding the use of ATR inhibitors in cancer chemotherapy regimens.Ultraviolet (UV) photoproducts and other bulky DNA lesions block the progression of DNA and RNA polymerases and lead to the activation of various DNA damage responses that are regulated by the action of the ataxia telangiectasia and Rad-3-related (ATR) 3 protein kinase. For example, in response to DNA polymerase stalling and uncoupling from DNA helicase activity (1), ATR is thought to be recruited to singlestranded regions of DNA where it promotes a number of DNA metabolic processes that maintain genomic integrity (2-6), including replication fork stabilization, DNA synthesis and lesion bypass, homologous recombination, replication origin firing, and cell cycle delay. The importance of ATR kinase activity in these responses in replicating cells is evidenced by the fate of cells in which the catalytic activity of ATR has been genetically or pharmacologically inhibited following replication stress, which include apoptosis and entry into catastrophic mitoses (7-10). Thus, ATR helps replicating cells cope with DNA damage and replication stress by facilitating cellular processes that maintain genomic stability and avoid cell death. Given these features of ATR, there has been great interest in the development and use of selective small-molecule inhibitors of ATR kinase activity in cancer chemotherapy regimens in conjunction with compounds that damage DNA and generate replication stress (11-13). For example, several studies have shown that ATR inhibition sensitizes cancer cells to cell kill...

“…Inhibition of Gap Filling Enhances DNA Damage Checkpoint Signaling and Induces Cell Death-Inhibition of gap-filling synthesis has been reported to lead to enlargement of excision gaps in human cells (39) and to enhanced DNA damage checkpoint signaling by the ATR kinase (40,41), which likely occurs through the recruitment of the ATR kinase to RPA-coated ssDNA excision gaps (42)(43)(44)(45). To confirm these findings and validate our experimental conditions, we therefore monitored the phosphorylation status of a number of protein substrates of the ATR kinase in UV-irradiated quiescent cells in the absence or presence of HU/AraC to limit gap filling.…”

Section: Primary and Partially Degraded Seddnas Are Differentially Exmentioning

confidence: 99%

DNA Repair Synthesis and Ligation Affect the Processing of Excised Oligonucleotides Generated by Human Nucleotide Excision Repair

Kemp

Gaddameedhi

Choi

et al. 2014

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Background:The mechanism of excised oligonucleotide processing during nucleotide excision repair is unknown. Results: UV photoproduct-containing oligonucleotides associate with chromatin following the dual incisions. Inhibition of gap-filling activities results in an accumulation of RPA-bound small, excised, damaged DNA (sedDNA) fragments. Conclusion: Gap filling-mediated dissociation of sedDNAs from RPA influences nucleotide excision repair rate. Significance: sedDNA processing is important in the DNA damage response.