In vivo measurements of interindividual differences in DNA glycosylases and APE1 activities

Chaim, Isaac A.; Nagel, Zachary D.; Jordan, Jennifer; Mazzucato, Patrizia; Ngo, Le P.; Samson, Leona D.

doi:10.1073/pnas.1712032114

Cited by 54 publications

(64 citation statements)

References 67 publications

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“…To more comprehensively determine the DNA repair capacity in the absence of CHD6, we used fluorescence multiplex host cell reactivation (FM-HCR) assays to measure: (i) non-homologous end-joining (NHEJ)-mediated DSB repair, (ii) homologous recombination (HR)-mediated DSB repair, (iii) nucleotide excision repair (NER) of ultraviolet C-induced DNA lesions, (iv) mismatch repair (MMR) of a G:G mispair and (v) BER of a tetrahydrofuran abasic site analog. In brief, fluorescent reporter plasmid substrates containing controlled quantities of each specific lesion are transfected into cells, whereupon DNA repair alters fluorescent reporter signal detectable by flow cytometry; these assays have been validated previously 36–38 . CHD6 deletion did not significantly impact NHEJ or HR-mediated DSB repair or BER pathways requiring SSB repair (Fig.…”

Section: Resultsmentioning

confidence: 99%

The CHD6 chromatin remodeler is an oxidative DNA damage response factor

et al. 2019

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Cell survival after oxidative DNA damage requires signaling, repair and transcriptional events often enabled by nucleosome displacement, exchange or removal by chromatin remodeling enzymes. Here, we show that Chromodomain Helicase DNA-binding protein 6 (CHD6), distinct to other CHD enzymes, is stabilized during oxidative stress via reduced degradation. CHD6 relocates rapidly to DNA damage in a manner dependent upon oxidative lesions and a conserved N-terminal poly(ADP-ribose)-dependent recruitment motif, with later retention requiring the double chromodomain and central core. CHD6 ablation increases reactive oxygen species persistence and impairs anti-oxidant transcriptional responses, leading to elevated DNA breakage and poly(ADP-ribose) induction that cannot be rescued by catalytic or double chromodomain mutants. Despite no overt epigenetic or DNA repair abnormalities, CHD6 loss leads to impaired cell survival after chronic oxidative stress, abnormal chromatin relaxation, amplified DNA damage signaling and checkpoint hypersensitivity. We suggest that CHD6 is a key regulator of the oxidative DNA damage response.

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Section: Resultsmentioning

confidence: 99%

The CHD6 chromatin remodeler is an oxidative DNA damage response factor

et al. 2019

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“…Given the BER defects observed through expression, localization, and response to induced DNA damage, we utilized a flow cytometric host cell reactivation assay (FM-HCR) to assess the repair capacities of TNBC cells across BER substrates [23, 24]. The FM-HCR assays measured DNA repair capacity using transiently transfected fluorescent reporter plasmids.…”

Section: Resultsmentioning

confidence: 99%

“…Fluorescence multiplex host cell reactivation (FM-HCR) assays were performed as described previously [23, 24]. Cells were seeded 48 hours before transfection into T25 tissue culture flasks (Thermofisher,156367) and collected for transfection at 85% confluence.…”

Section: Methodsmentioning

confidence: 99%

“…Reporter expression was reported as a percentage of fluorescent reporter protein expression from undamaged control plasmids in a second transfection that was also normalized for transfection efficiency. Fluorescent signal generated from the repair of the damaged and undamaged substrates was quantitated in each triplicate, and percent reporter expression was calculated as described previously [23, 24].…”

Section: Methodsmentioning

confidence: 99%

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Defective base excision repair in the response to DNA damaging agents in triple negative breast cancer

Lee

Piett

Andrews

et al. 2019

Preprint

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21 22 23DNA repair defects have been increasingly focused on as therapeutic targets. In hormone 24 positive breast cancer, XRCC1-deficient tumors have been identified and proposed as 25 targets for combination therapies that damage DNA and inhibit DNA repair pathways. 26XRCC1 is a scaffold protein that functions in base excision repair (BER) by mediating 27 essential interactions between DNA glycosylases, AP endonuclease, poly(ADP-ribose) 28 polymerase 1, DNA polymerase β (POL β), and DNA ligases. Loss of XRCC1 confers 29 BER defects and hypersensitivity to DNA damaging agents. BER defects have not been 30 evaluated in triple negative breast cancer (TNBC), for which new therapeutic targets and 31 therapies are needed. To evaluate the potential of XRCC1 as an indicator of BER defects 32 in TNBC, we examined XRCC1 expression and localization in the TCGA database and in 33 TNBC cell lines. High XRCC1 expression was observed for TNBC tumors in the TCGA 34 database and expression of XRCC1 varied between TNBC cell lines. We also observed 35 changes in XRCC1 subcellular localization in TNBCs that alter the ability to repair base 36 lesions and single-strand breaks. Subcellular localization changes were also observed for 37 POL β that did not correlate with XRCC1 localization. Basal levels of DNA damage were 38 also measured in the TNBC cell lines, and damage levels correlated with observed 39 changes in XRCC1 expression, localization, and repair functions. The results confirmed 40 that XRCC1 expression changes may indicate DNA repair capacity changes but 41 emphasize that basal DNA damage levels along with expression and localization are 42 better indicators of DNA repair defects. Given the observed over-expression of XRCC1 in 43 TNBC preclinical models and the TCGA database, XRCC1 expression levels should be 44 considered when evaluating treatment responses of TNBC preclinical model cells. 45 3 Keywords: triple negative breast cancer, base excision repair, XRCC1, PARP1, DNA 46 repair, DNA damage, nuclear localization 47 48 Defects in DNA damage response and repair are driving factors in carcinogenesis 49and key determinants in the response to chemotherapy. Breast cancers may display 50 defects in DNA repair such as mutations in key DNA damage response and repair 51 proteins such as breast cancer-susceptibility (BRCA1/2) and tumor suppressor protein 52 p53 (TP53), and altered expression levels of DNA repair proteins thymine-DNA 53 glycosylase (TDG) and poly(ADP-ribose) polymerase 1 (PARP1) [1-3]. Therapeutic 54 outcomes may be improved by exploiting DNA repair defects present in cancer cells but 55 absent in normal cells, as in the use of PARP-inhibitors (PARPi) in cancers that have 56 BRCA1/2 deficiencies. However, characterization of DNA repair pathways often is lacking 57 in preclinical models and cell lines. Examining DNA repair defects in preclinical models 58 and patients is essential for evaluating the efficacy of therapeutic agents. 59 In breast cancer, DNA repair defects often extend beyond homologous 60 re...

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“…Here, we tested the Q205* and A207P mutants, which yield apurinic lesions (in contrast to physiologically less relevant apyrimidinic sites), in HeLa cells. Considering that the transcriptional bypass of AP lesions in human cells occurs predominantly with incorporation of A [ 64 ], the resulting base substitutions should be detectable in both Q205* and A207P reporters ( Figure 3 ). Despite the NER proficiency of the host cell line, we detected a signal at the level of >30% wild-type EGFP in both reporter constructs ( Figure 1 b and Figure 4 c), which indicates extremely high detection sensitivity of TM.…”

Section: Discussionmentioning

confidence: 99%

EGFP Reporters for Direct and Sensitive Detection of Mutagenic Bypass of DNA Lesions

2020

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The sustainment of replication and transcription of damaged DNA is essential for cell survival under genotoxic stress; however, the damage tolerance of these key cellular functions comes at the expense of fidelity. Thus, translesion DNA synthesis (TLS) over damaged nucleotides is a major source of point mutations found in cancers; whereas erroneous bypass of damage by RNA polymerases may contribute to cancer and other diseases by driving accumulation of proteins with aberrant structure and function in a process termed “transcriptional mutagenesis” (TM). Here, we aimed at the generation of reporters suited for direct detection of miscoding capacities of defined types of DNA modifications during translesion DNA or RNA synthesis in human cells. We performed a systematic phenotypic screen of 25 non-synonymous base substitutions in a DNA sequence encoding a functionally important region of the enhanced green fluorescent protein (EGFP). This led to the identification of four loss-of-fluorescence mutants, in which any ulterior base substitution at the nucleotide affected by the primary mutation leads to the reversal to a functional EGFP. Finally, we incorporated highly mutagenic abasic DNA lesions at the positions of primary mutations and demonstrated a high sensitivity of detection of the mutagenic DNA TLS and TM in this system.

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In vivo measurements of interindividual differences in DNA glycosylases and APE1 activities

Cited by 54 publications

References 67 publications

The CHD6 chromatin remodeler is an oxidative DNA damage response factor

The CHD6 chromatin remodeler is an oxidative DNA damage response factor

Defective base excision repair in the response to DNA damaging agents in triple negative breast cancer

EGFP Reporters for Direct and Sensitive Detection of Mutagenic Bypass of DNA Lesions

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