A Quantitative Mass Spectrometry-Based Approach for Assessing the Repair of 8-Methoxypsoralen-Induced DNA Interstrand Cross-Links and Monoadducts in Mammalian Cells

Liu, Shuo; Wang, Yinsheng

doi:10.1021/ac4012232

Cited by 21 publications

(17 citation statements)

References 50 publications

(107 reference statements)

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“…Thus it was desirable to have a more direct assessment of the ICLs embedded in the replication tracts. It has been shown by LC-MS/MS that psoralen monoadducts are not removed in ERCC1 −/ − cells (Liu and Wang, 2013). If, during replication, there had been an appreciable unhooking of ICLs (Sengerova et al, 2012; Smeaton et al, 2008), there would be a decline in the ICL: MA ratio in the DNA in the replication tracts, relative to the ratio in DNA harvested from cells immediately after treatment.…”

Section: Resultsmentioning

confidence: 99%

The DNA Translocase FANCM/MHF Promotes Replication Traverse of DNA Interstrand Crosslinks

et al. 2013

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SUMMARY The replicative machinery encounters many impediments, some of which can be overcome by lesion bypass or replication restart pathways, leaving repair for a later time. However, interstrand crosslinks (ICLs), which preclude DNA unwinding, are considered absolute blocks to replication. Current models suggest that fork collisions, either from one or both sides of an ICL, initiate repair processes required for resumption of replication. To test these proposals, we developed a single molecule technique for visualizing encounters of replication forks with ICLs, as they occur in living cells. Surprisingly, the most frequent patterns were consistent with replication traverse of an ICL, without lesion repair. The traverse frequency was strongly reduced by inactivation of the translocase and DNA binding activities of the FANCM/MHF complex. The results indicate that translocase-based mechanisms enable DNA synthesis to continue past ICLs, and that these lesions are not always absolute blocks to replication.

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Section: Resultsmentioning

confidence: 99%

The DNA Translocase FANCM/MHF Promotes Replication Traverse of DNA Interstrand Crosslinks

et al. 2013

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“…To improve the separation and detection of DNA ICLs introduced by nitrogen mustard melphalan, Mohamed and Linscheid 165 used a combination of benzonase and nuclease S1, together with careful control of the digestion time, to release the cross-link moiety as a trinucleotide. Wang and co-workers 4,92,166,167 also employed nuclease P1 to degrade ICL-containing DNA induced by psoralen derivatives to a tetranucleotide remnant for LC-MS and MS/MS analyses (Fig. 3a).…”

Section: Quantitative Analysis Of Dna Adductsmentioning

confidence: 99%

“…By using HPLC-ESI-MS/MS, Liu et al 167 recently studied the repair of DNA ICL and MAs introduced by 8-MOP in cultured mammalian cells. The detection limit was found to be as low as 3 fmol, which facilitated the observation of a rapid clearance of 8-MOP-ICL from cellular DNA 24 h post-irradiation.…”

Section: Quantitative Analysis Of Dna Adductsmentioning

confidence: 99%

Mass spectrometry for the assessment of the occurrence and biological consequences of DNA adducts

2015

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Exogenous and endogenous sources of chemical species can react, directly or after metabolic activation, with DNA to yield DNA adducts. If not repaired, DNA adducts may compromise cellular functions by blocking DNA replication and/or inducing mutations. Unambiguous identification of the structures and accurate measurements of the levels of DNA adducts in cellular and tissue DNA constitute the first and important step towards understanding the biological consequences of these adducts. The advances in mass spectrometry (MS) instrumentation in the past 2–3 decades have rendered MS an important tool for structure elucidation, quantification, and revelation of the biological consequences of DNA adducts. In this review, we summarized the development of MS techniques on these fronts for DNA adduct analysis. We placed our emphasis of discussion on sample preparation, the combination of MS with gas chromatography-or liquid chromatography (LC)-based separation techniques for the quantitative measurement of DNA adducts, and the use of LC-MS along with molecular biology tools for understanding the human health consequences of DNA adducts. The applications of mass spectrometry-based DNA adduct analysis for predicting the therapeutic outcome of anti-cancer agents, for monitoring the human exposure to endogenous and environmental genotoxic agents, and for DNA repair studies were also discussed.

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“…Since the first application of mass spectrometry techniques in nucleic acid research, HPLC coupled with tandem mass spectrometry (LC-MS/MS) has become one of the standard methods for quantifying DNA modifications [22]. For instance, an LC-MS/MS coupled with the stable isotope-dilution method allowed for the examination of the roles of repair proteins in removing bulky DNA lesions from mammalian genome [23]. It has also been extensively used for the analysis of 5-mC and its oxidation products, which may be involved in epigenetic regulation of a broad range of biological processes and diseases [24].…”

Section: Introductionmentioning

confidence: 99%

Quantitative Mass Spectrometry-Based Analysis of β-D-Glucosyl-5-Hydroxymethyluracil in Genomic DNA of Trypanosoma brucei

Liu

Cliffe

et al. 2014

J. Am. Soc. Mass Spectrom.

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β-D-glucosyl-5-hydroxymethyluracil (base J) is a hyper-modified nucleobase found in the nuclear DNA of kinetoplastid parasites. With replacement of a fraction of thymine in DNA, J is localized primarily in telomeric regions of all organisms carrying this modified base. The biosynthesis of J occurs in two putative steps: First, a specific thymine in DNA is recognized and converted into 5-hydroxymethyluracil (5-HmU) by J-binding proteins (JBP1 and JBP2); a glucosyl transferase (GT) subsequently glucosylates the 5-HmU to yield J. Although several recent studies revealed the roles of internal J in regulating transcription in kinetoplastids, functions of telomeric J and proteins involved in J synthesis remain elusive. Assessing the functions of base J and understanding fully its biosynthesis necessitate the measurement of its level in cells and organisms. In this study, we reported a reversed-phase HPLC coupled with tandem mass spectrometry (LC-MS/MS) method, together with the use of a surrogate internal standard (β-D-glucosyl-5-hydroxymethyl-2′-deoxycytidine, 5-gHmdC), for the accurate detection of β-D-glucosyl-5-hydroxymethyl-2′-deoxyuridine (dJ) in Trypanosoma brucei DNA. For comparison, we also measured the level of the precursor for dJ synthesis, i.e. 5-hydroxymethyl-2′-deoxyuridine (5-HmdU). We found that base J was not detectable in the JBP-null cells while it replaced approximately 0.5% thymine in wild-type cells, which was accompanied with a markedly decreased level of 5-HmdU in JBP1/JBP2-null strain relative to the wild-type strain. These results provided direct evidence supporting that JBP proteins play an important role in oxidizing thymidine to form 5-HmdU, which facilitated the generation of dJ. This is the first report about the application of LC-MS/MS for the quantification of base J. The analytical method built a solid foundation for dissecting the molecular mechanisms of J biosynthesis and assessing the biological functions of base J in the future.

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A Quantitative Mass Spectrometry-Based Approach for Assessing the Repair of 8-Methoxypsoralen-Induced DNA Interstrand Cross-Links and Monoadducts in Mammalian Cells

Cited by 21 publications

References 50 publications

The DNA Translocase FANCM/MHF Promotes Replication Traverse of DNA Interstrand Crosslinks

The DNA Translocase FANCM/MHF Promotes Replication Traverse of DNA Interstrand Crosslinks

Mass spectrometry for the assessment of the occurrence and biological consequences of DNA adducts

Quantitative Mass Spectrometry-Based Analysis of β-D-Glucosyl-5-Hydroxymethyluracil in Genomic DNA of Trypanosoma brucei

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