Quantification of DNA Damage Using Mass Spectrometry Techniques

2014 ◽  
pp. 203-224
Author(s):  
Thierry Douki ◽  
Jean-Luc Ravanat
Keyword(s):  
Mass Spectrometry ◽  
Dna Damage

scholarly journals Interaction of Deubiquitinase 2A-DUB/MYSM1 with DNA Repair and Replication Factors

2020 ◽  
Vol 21 (11) ◽  
pp. 3762
Author(s):  
Carsten Kroeger ◽  
Reinhild Roesler ◽  
Sebastian Wiese ◽  
Adelheid Hainzl ◽  
Martina Vanessa Gatzka
Keyword(s):  
Mass Spectrometry ◽  
Dna Damage ◽  
Dna Repair ◽  
Transcriptional Activation ◽  
Mononuclear Cells ◽  
Bone Marrow Cells ◽  
Human Peripheral Blood ◽  
Cell Cycle Control ◽  
Mass Spectrometry Data ◽  
Peripheral Blood Mononuclear

The deubiquitination of histone H2A on lysine 119 by 2A-DUB/MYSM1, BAP1, USP16, and other enzymes is required for key cellular processes, including transcriptional activation, apoptosis, and cell cycle control, during normal hematopoiesis and tissue development, and in tumor cells. Based on our finding that MYSM1 colocalizes with γH2AX foci in human peripheral blood mononuclear cells, leukemia cells, and melanoma cells upon induction of DNA double-strand breaks with topoisomerase inhibitor etoposide, we applied a mass spectrometry-based proteomics approach to identify novel 2A-DUB/MYSM1 interaction partners in DNA-damage responses. Differential display of MYSM1 binding proteins significantly enriched after exposure of 293T cells to etoposide revealed an interacting network of proteins involved in DNA damage and replication, including factors associated with poor melanoma outcome. In the context of increased DNA-damage in a variety of cell types in Mysm1-deficient mice, in bone marrow cells upon aging and in UV-exposed Mysm1-deficient skin, our current mass spectrometry data provide additional evidence for an interaction between MYSM1 and key DNA replication and repair factors, and indicate a potential function of 2A-DUB/MYSM1 in DNA repair processes.

scholarly journals Capturing RNA–protein interaction via CRUIS

2020 ◽  
Vol 48 (9) ◽  
pp. e52-e52 ◽  
Author(s):  
Ziheng Zhang ◽  
Weiping Sun ◽  
Tiezhu Shi ◽  
Pengfei Lu ◽  
Min Zhuang ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Dna Damage ◽  
Protein Interactions ◽  
Noncoding Rna ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Living Cells ◽  
Innovative Methods ◽  
Rna Biology

Abstract No RNA is completely naked from birth to death. RNAs function with and are regulated by a range of proteins that bind to them. Therefore, the development of innovative methods for studying RNA–protein interactions is very important. Here, we developed a new tool, the CRISPR-based RNA-United Interacting System (CRUIS), which captures RNA–protein interactions in living cells by combining the power of CRISPR and PUP-IT, a novel proximity targeting system. In CRUIS, dCas13a is used as a tracker to target specific RNAs, while proximity enzyme PafA is fused to dCas13a to label the surrounding RNA-binding proteins, which are then identified by mass spectrometry. To identify the efficiency of CRUIS, we employed NORAD (Noncoding RNA activated by DNA damage) as a target, and the results show that a similar interactome profile of NORAD can be obtained as by using CLIP (crosslinking and immunoprecipitation)-based methods. Importantly, several novel NORAD RNA-binding proteins were also identified by CRUIS. The use of CRUIS facilitates the study of RNA–protein interactions in their natural environment, and provides new insights into RNA biology.

Physiological evidence of DNA damage by carcinogens known to be present in charred and processed meats (PhIP DNA adducts) in a small cohort of patients with prostate cancer.

2017 ◽  
Vol 35 (6_suppl) ◽  
pp. e580-e580
Author(s):  
Christopher J. Weight ◽  
Shun Xiao ◽  
Jingshu Guo ◽  
Byeong Hwa ◽  
Peter Villalta ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Mass Spectrometry ◽  
Dna Damage ◽  
Dna Adducts ◽  
Dna Adduct ◽  
Spectrometry Method ◽  
Mass Spectrometry Method ◽  
Processed Meats ◽  
Tumor Bearing ◽  
Ffpe Tissues

e580 Background: Epidemiologic studies have reported an association between frequent consumption of well-done cooked meats and prostate cancer (PC) risk. Charred red meat and cooked processed meats are known to contain heterocyclic aromatic amine (HAA) carcinogens, such as 2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine (PhIP) the most mass abundant HAA, and are linked to PC development in a rodent model. However, unambiguous physiochemical markers of DNA damage from these meat-derived carcinogens have not been identified in human samples to support the paradigm of HAA induced human prostate carcinogenesis. Methods: Thirty-five men with biopsy proven intermediate to high-risk PC underwent radical prostatectomy at University of Minnesota from Dec 2015-Aug 2016. After prostatectomy, both tumor bearing tissue and non-tumor bearing adjacent fresh tissue was analyzed for DNA adducts using a highly sensitive nano-LC-Orbitrap mass spectrometry method. We also analyzed formalin fixed paraffin embedded (FFPE) tissues from each patient. Results: Median age of the men with PC was 65 (range 45-78). Pathology demonstrated the following Gleason Scores (GS) and pathologic staging: GS = 6 in 1 patient (2.8%), GS = 7 in 28 patients (80%) and GS = 8-10 in 6 patients (17%) and 16 men (46%) were stage 2 and 19 men were stage 3 (54%). The PhIP DNA adduct was identified in 11 out of 35 patients, at levels ranging from 2 to 120 adducts per 109 nucleotides. PhIP DNA adducts also were recovered quantitatively from FFPE tissues. Conclusions: Our data provide support to the epidemiological observations implicating PhIP as a DNA damaging agent that may contribute to the etiology of PC in humans. FFPE tissues can be used as a tissue source in DNA-adduct biomarker research using our mass spectrometry method

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