Slow repair of lipid peroxidation-induced DNA damage at p53 mutation hotspots in human cells caused by low turnover of a DNA glycosylase

Woodrick, Jordan; Gupta, Suhani; Khatkar, Pooja; Sarangi, Sanchita; Narasimhan, Ganga; Trehan, Akriti; Adhikari, Sanjay; Roy, Rabindra

doi:10.1093/nar/gku520

Cited by 9 publications

(11 citation statements)

References 49 publications

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“…In the case of APE1, functional activity of the enzyme on the AP-site will result in complete repair of the DNA. We modified and simplified a previously described real time PCR-based method for assaying repair in cells to monitor repair of the engineered AP-site phagemid DNA (15,16,19). The strategy for the real time PCR-based AP-site repair assay is depicted in Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The real time PCR-based assay for repair was performed and analyzed as previously described with some modification and simplification (16,19). The control region was amplified using the following primers: forward 5′-GTGACGAAACTCAGTGTTAC-3′ and reverse 5′-TCGAGAGGGTTGATATAAG-3′.…”

Section: Methodsmentioning

confidence: 99%

“…We further validated this method by monitoring the recruitment of downstream BER proteins, including Polβ, LIG1, and FEN1, in a lesion-specific manner. Finally, we determined the functionality of the observed BER enzyme-lesion DNA co-localization by modifying some in vivo DNA repair assays developed previously by our lab (15,16). …”

Section: Introductionmentioning

confidence: 99%

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A novel method for monitoring functional lesion-specific recruitment of repair proteins in live cells

Woodrick

Gupta

Khatkar

et al. 2015

Mutation Research/Fundamental and Molecular Mechanisms of Mutag

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DNA-protein relationships have been studied by numerous methods, but a particular gap in methodology lies in the study of DNA adduct-specific interactions with proteins in vivo, which particularly affects the field of DNA repair. Using the repair of a well-characterized and ubiquitous adduct, the abasic (AP) site, as a model, we have developed a comprehensive method of monitoring DNA lesion-specific recruitment of proteins in vivo over time. We utilized a surrogate system in which a Cy3-labeled plasmid containing a single AP-site was transfected into cells, and the interaction of the labeled DNA with BER enzymes, including APE1, Polβ, LIG1, and FEN1, was monitored by immunofluorescent staining of the enzymes by Alexafluor-488-conjugated secondary antibody. The recruitment of enzymes was characterized by quantification of Cy3-Alexafluor-488 co-localization. To validate the microscopy-based method, repair of the transfected AP-site DNA was also quantified at various time points post-transfection using a real time PCR-based method. Notably, the recruitment time kinetics for each enzyme were consistent with AP-site repair time kinetics. This microscopy-based methodology is reliable in detecting the recruitment of proteins to specific DNA substrates and can be extended to study other in vivo DNA-protein relationships in any DNA sequence and in the context of any DNA structure in transfectable proliferating or quiescent cells. The method may be applied to a variety of disciplines of nucleic acid transaction pathways, including repair, replication, transcription, and recombination.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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A novel method for monitoring functional lesion-specific recruitment of repair proteins in live cells

Woodrick

Gupta

Khatkar

et al. 2015

Mutation Research/Fundamental and Molecular Mechanisms of Mutag

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“…A repair polymerase, DNA polymerase β fills in the nucleotide gap and removes the dRP. Finally, a DNA ligase seals the nick and completes Hx repair [10,11]. …”

Section: Introductionmentioning

confidence: 99%

Mutagenic potential of hypoxanthine in live human cells

Devito

Woodrick

Song

et al. 2017

Mutation Research/Fundamental and Molecular Mechanisms of Mutag

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Hypoxanthine (Hx) is a major DNA lesion generated by deamination of adenine during chronic inflammatory conditions, which is an underlying cause of various diseases including cancer of colon, liver, pancreas, bladder and stomach. There is evidence that deamination of DNA bases induces mutations, but no study has directly linked Hx accumulation to mutagenesis and strand-specific mutations yet in human cells. Using a site-specific mutagenesis approach, we report the first direct evidence of mutation potential and pattern of Hx in live human cells. We investigated Hx-induced mutations in human nonmalignant HEK293 and cancer HCT116 cell lines and found that Hx is mutagenic in both HEK293 and HCT116 cell lines. There is a strand bias for Hx-mediated mutations in both the cell lines; the Hx in lagging strand is more mutagenic than in leading strand. There is also some difference in cell types regarding the strand bias for mutation types; HEK293 cells showed largely deletion (>80%) mutations in both leading and lagging strand and the rest were insertions and A:T→G:C transition mutations in leading and lagging strands, respectively, whereas in HCT116 cells we observed 60% A:T→G:C transition mutations in the leading strand and 100% deletions in the lagging strand. Overall, Hx is a highly mutagenic lesion capable of generating A:T→G:C transitions and large deletions with a significant variation in leading and lagging strands in human cells. In recent meta-analysis study A→G (T→C) mutations were found to be a prominent signature in a variety of cancers, including a majority types that are induced by inflammation. The deletions are known to be a major cause of copy-number variations or CNVs, which is a major underlying cause of many human diseases including mental illness, developmental disorders and cancer. Thus, Hx, a major DNA lesion induced by different deamination mechanisms, has potential to initiate inflammation-driven carcinogenesis in addition to various human pathophysiological consequences.

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“…Real time PCR has been widely used in many fields including DNA and RNA quantitation, genotyping studies, mutation analysis, and genetic diagnosis, [1][2][3][4][5] since it can monitor the reaction product in real time, provide accurate quantification in a widely dynamic range, and more importantly eliminate the need for post-reaction gel analysis. Many of these applications increasingly require high-throughput experiments in real time PCR using 96-or 384-well plates that may have a large well-towell variation arising from the time lag during the mixing and dispensing steps.…”

Section: Introductionmentioning

confidence: 99%

Development of a high-throughput real time PCR based on a hot-start alternative for Pfu mediated by quantum dots

et al. 2015

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Hot start (HS) PCR is an excellent alternative for high-throughput real time PCR due to its ability to prevent nonspecific amplification at low temperature. Development of a cost-effective and simple HS PCR technique to guarantee high-throughput PCR specificity and consistency still remains a great challenge. In this study, we systematically investigated the HS characteristics of QDs triggered in real time PCR with EvaGreen and SYBR Green I dyes by the analysis of amplification curves, standard curves and melting curves. Two different kinds of DNA polymerases, Pfu and Taq, were employed. Here we showed that high specificity and efficiency of real time PCR were obtained in a plasmid DNA and an error-prone two-round PCR assay using QD-based HS PCR, even after an hour preincubation at 50 °C before real time PCR. Moreover, the results obtained by QD-based HS PCR were comparable to a commercial Taq antibody DNA polymerase. However, no obvious HS effect of QDs was found in real time PCR using Taq DNA polymerase. The findings of this study demonstrated that a cost-effective high-throughput real time PCR based on QD triggered HS PCR could be established with high consistency, sensitivity and accuracy.

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Slow repair of lipid peroxidation-induced DNA damage at p53 mutation hotspots in human cells caused by low turnover of a DNA glycosylase

Cited by 9 publications

References 49 publications

A novel method for monitoring functional lesion-specific recruitment of repair proteins in live cells

A novel method for monitoring functional lesion-specific recruitment of repair proteins in live cells

Mutagenic potential of hypoxanthine in live human cells

Development of a high-throughput real time PCR based on a hot-start alternative for Pfu mediated by quantum dots

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