Quantitative Measurement of Translesion DNA Synthesis in Mammalian Cells

Ziv, Omer; Diamant, Noam; Shachar, Sigal; Hendel, Ayal; Livneh, Zvi

doi:10.1007/978-1-61779-998-3_35

Cited by 9 publications

(12 citation statements)

References 19 publications

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“…Exclusion of false-positive hits due to off-target effects of the siRNA was achieved by de-convolution of selected siRNA pools and testing each of the four siRNA oligos separately. Gap-lesion plasmids were constructed as described 66 .…”

Section: Methodsmentioning

confidence: 99%

Identification of novel DNA-damage tolerance genes reveals regulation of translesion DNA synthesis by nucleophosmin

et al. 2014

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Cells cope with replication-blocking lesions via translesion DNA synthesis (TLS). TLS is carried out by low-fidelity DNA polymerases that replicate across lesions, thereby preventing genome instability at the cost of increased point mutations. Here we perform a two-stage siRNA-based functional screen for mammalian TLS genes and identify 17 validated TLS genes. One of the genes, NPM1, is frequently mutated in acute myeloid leukaemia (AML). We show that NPM1 (nucleophosmin) regulates TLS via interaction with the catalytic core of DNA polymerase-η (polη), and that NPM1 deficiency causes a TLS defect due to proteasomal degradation of polη. Moreover, the prevalent NPM1c+ mutation that causes NPM1 mislocalization in ~30% of AML patients results in excessive degradation of polη. These results establish the role of NPM1 as a key TLS regulator, and suggest a mechanism for the better prognosis of AML patients carrying mutations in NPM1.

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Section: Methodsmentioning

confidence: 99%

Identification of novel DNA-damage tolerance genes reveals regulation of translesion DNA synthesis by nucleophosmin

et al. 2014

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“…1,3,4,9,10,31 These vector-based model systems have provided a plethora of useful information for gauging the blocking and mutagenic potentials of DNA lesions in cells. However, lesion-induced changes in genetic information revealed by these vector-based studies may differ, to some extent, from their effects on replication or transcription when they are located on chromosomes in cells.…”

Section: Discussionmentioning

confidence: 99%

Mass Spectrometry-Based Quantitative Strategies for Assessing the Biological Consequences and Repair of DNA Adducts

You

Wang

2016

Acc. Chem. Res.

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The genetic integrity of living organisms is constantly threatened by environmental and endogenous sources of DNA damaging agents that can induce a plethora of chemically modified DNA lesions. Unrepaired DNA lesions may elicit cytotoxic and mutagenic effects and contribute to the development of human diseases including cancer and neurodegeneration. Understanding the deleterious outcomes of DNA damage necessitates the investigation about the effects of DNA adducts on the efficiency and fidelity of DNA replication and transcription. Conventional methods for measuring lesion-induced replicative or transcriptional alterations often require time-consuming colony screening and DNA sequencing procedures. Recently, a series of mass spectrometry (MS)-based strategies have been developed in our laboratory as an efficient platform for qualitative and quantitative analyses of the changes in genetic information induced by DNA adducts during DNA replication and transcription. During the past few years, our laboratory has used these MS-based methods for assessing the replicative or transcriptional blocking and miscoding properties of more than 30 distinct DNA adducts. When combined with genetic manipulation, these methods have also been successfully employed for revealing the roles of various DNA repair proteins or translesion synthesis DNA polymerases in modulating the adverse effects of DNA lesions on transcription or replication in mammalian and bacterial cells. In this Account, we focus on the development of MS-based approaches for determining the effects of DNA adducts on DNA replication and transcription in vitro as well as in bacterial and mammalian cells. We also highlight their applications to lesion bypass, mutagenesis and repair studies of three representative types of DNA lesions, i.e., methylglyoxal-induced N2-(1-carboxyethyl)-2'-deoxyguanosine (N2-CEdG), oxidatively induced 8,5'-cyclopurine-2'-deoxynucleosides, and regioisomeric alkylated thymidine lesions. Specially, we discuss the similar and distinct effects of the minor-groove DNA lesions including N2-CEdG and O2-alkylated thymidine lesions as well as the major-groove O4-alkylated thymidine lesions on DNA replication and transcription machinery. The MS-based strategies described herein should be generally applicable for quantitative measurement of the biological consequences and repair of other DNA lesions in vitro and in cells.

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“…The construction of the control gapped plasmid GP20 was previously described [21–23]. A gap-lesion plasmid with a site-specific TT6-4PP flanked by sequences used previously in the gap-lesion plasmid system, was prepared using the site-specifically modified 11-mer oligonucleotide 5′-GCAAG TT GGAG-3′ containing a single site-specific TT 6-4 PP (underlined) as previously described [19].…”

Section: Methodsmentioning

confidence: 99%

“…The TLS assay was executed as previously described [23,25], using SV40-transformed MRC5sv human fetal lung fibroblasts [27] or mouse embryo fibroblasts [26]. Briefly, transfection was done in 6-well plates (surface area 9.1 cm 2 ) at ~80% confluence using Jet-PEI transfection reagent.…”

Section: Methodsmentioning

confidence: 99%

DNA sequence context greatly affects the accuracy of bypass across an ultraviolet light 6-4 photoproduct in mammalian cells

Shriber

Leitner‐Dagan

Geacintov

et al. 2015

Mutation Research/Fundamental and Molecular Mechanisms of Mutag

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Translesion DNA synthesis (TLS) is a DNA damage tolerance mechanism carried out by low-fidelity DNA polymerases that bypass DNA lesions, which overcomes replication stalling. Despite the miscoding nature of most common DNA lesions, several of them are bypassed in mammalian cells in a relatively accurate manner, which plays a key role maintaining a low mutation load. Whereas it is generally agreed that TLS across the major UV and sunlight induced DNA lesion, the cyclobutane pyrimidine dimer (CPD), is accurate, there were conflicting reports on whether the same is true for the thymine–thymine pyrimidine–pyrimidone(6-4) ultraviolet light photoproduct (TT6-4PP), which represents the second most common class of UV lesions. Using a TLS assay system based on gapped plasmids carrying site-specific TT6-4PP lesions in defined sequence contexts we show that the DNA sequence context markedly affected both the extent and accuracy of TLS. The sequence exhibiting higher TLS exhibited also higher error-frequency, caused primarily by semi-targeted mutations, at the nearest nucleotides flanking the lesion. Our results resolve the discrepancy reported on TLS across TT6-4PP, and suggest that TLS is more accurate in human cells than in mouse cells.

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Quantitative Measurement of Translesion DNA Synthesis in Mammalian Cells

Cited by 9 publications

References 19 publications

Identification of novel DNA-damage tolerance genes reveals regulation of translesion DNA synthesis by nucleophosmin

Identification of novel DNA-damage tolerance genes reveals regulation of translesion DNA synthesis by nucleophosmin

Mass Spectrometry-Based Quantitative Strategies for Assessing the Biological Consequences and Repair of DNA Adducts

DNA sequence context greatly affects the accuracy of bypass across an ultraviolet light 6-4 photoproduct in mammalian cells

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