紫外线辐射处理后DNA纤维的检测

Alfano  Luigi; Antonio  Giordano; Francesca  Pentimalli

doi:10.21769/BioProtoc.2301

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Peer-reviewed

DNA Fiber Assay upon Treatment with Ultraviolet Radiations

紫外线辐射处理后DNA纤维的检测

Alfano Luigi

Antonio Giordano email

Francesca Pentimalli

发布: 2017年06月05日第7卷第11期 DOI: 10.21769/BioProtoc.2301 浏览次数: 21124

评审: Anonymous reviewer(s)

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Abstract

Genome stability is continuously challenged by a wide range of DNA damaging factors. To promote a correct DNA repair and cell survival, cells orchestrate a coordinated and finely tuned cascade of events collectively known as the DNA Damage Response (DDR). Ultra Violet (UV) rays are among the main environmental sources of DNA damage and a well recognized cancer risk factor. UV rays induce the formation of toxic cyclobutane-type pyrimidine dimers (CPD) and [6-4]pyrimidine-pyrimidone (6-4PP) photoproducts which trigger the activation of the intra-S phase cell cycle checkpoint (Kaufmann, 2010) aimed at preventing replication fork collapse, late origin firing, and stabilizing fragile sites (Branzei and Foiani, 2009). To monitor the activation of the intra-S phase checkpoint in response to UV type C (UVC) exposure, the DNA fiber assay can be used to analyse the new origin firing and DNA synthesis rate (Jackson et al., 1998; Merrick et al., 2004; Alfano et al., 2016). The DNA fiber assay technique was conceived in the 90s and then further developed through the use of thymidine analogues (such as CldU and IdU), which are incorporated into the nascent DNA strands. By treating the cells in sequential mode with these analogues, which can be visualized through specific antibodies carrying different fluorophores, it is possible to monitor the replication fork activity and assess how this is influenced by UV radiations or others agents.

Keywords: Ultra Violet radiation (紫外线辐射)

DNA damage (DNA损伤)

Cell cycle checkpoints (细胞周期检查点)

DNA fiber assay (DNA纤维检测试验)

Halogenated pyrimidines (卤代嘧啶)

Background

Genomic instability is one of the hallmarks of cancer involved in both tumour development and progression (Hanahan and Weinberg, 2011). The preservation of genomic stability depends on a complex cascade of finely tuned events which are collectively known as the DNA damage response. This includes the activation of cell cycle checkpoints, which stall cell cycle to allow the cellular repair machinery to mend the damage. The identification of novel druggable proteins, involved in the DNA repair mechanisms, is part of modern cancer therapy. In this context, the DNA fiber assay can be used as a readout of replication fork activity to identify new potential players in the regulation of the intra-S phase checkpoint, which is triggered upon exposure to various chemotherapeutic drugs.

The unique chemistry of DNA, of its constituents and its chemical-physical properties allowed pioneering scientists to visualize the whole length of DNA fibers, first by labelling DNA with tritiated thymidine followed by detection through autoradiography (Cairns, 1963). Then Bensimon and colleagues showed that it was possible to align and ‘comb’ the DNA fibers on a solid matrix achieving a uniform stretching and an easy access for specific hybridizations (Bensimon et al., 1994). Jackson and Pombo were the first to use sequential labelling with halogenated pyrimidines, such as bromo-deoxyuridine (BrdU) and iodo-deoxyuridine (IdU), which are incorporated into DNA as thymidine analogues (Jackson and Pombo, 1998). This allowed them to assess qualitatively and quantitatively the activity of replicon clusters in HeLa cells at different times during S phase. Afterwards another study (Merrick et al., 2004) described a modified DNA fiber labelling, which was adapted by a classical DNA fiber autoradiography (Huberman and Riggs, 1968). Diffley and colleagues used pulse labelling with two halogenated nucleotides: chloro-deoxyuridine (CldU) and IdU, which could be differentially detected through specific antibodies, each carrying a different fluorescent dye. This allowed to visualize through fluorescent microscopy the effect of a specific cell treatment on the dynamics of the DNA synthesis process. The protocol described below was made as described by the two previously cited protocols with some modifications (Jackson and Pombo, 1998; Merrick et al., 2004).

Materials and Reagents

60 mm cell culture dishes (Sigma-Aldrich, catalog number: CLS430166 )
Manufacturer: Corning, catalog number: 430166.
Silane-Prep Slides: glass slides coated with silane (aminoalkylsilane) (Sigma-Aldrich, catalog number: S4651 )
HeLa cells (ATCC, catalog number: CCL-2 )
Cell culture
1. Roswell Park Memorial Institute (RPMI) 1640 medium (Thermo Fisher Scientific, Gibco^TM, catalog number: 61870010 )
2. 10% fetal bovine serum (FBS) (Thermo Fisher Scientific, Gibco^TM, catalog number: 10270106 )
3. Penicillin-streptomycin (Thermo Fisher Scientific, Gibco^TM, catalog number: 15070063 )
4. 0.25% trypsin-EDTA (Thermo Fisher Scientific, catalog number: 25200056 )
Note: HeLa cells were cultured in RPMI 1640 supplemented with 10% FBS, 1 μg/ml penicillin and 1 µg/ml streptomycin (defined as ‘complete medium’).
5-Iodo-2’-deoxyuridine (IdU) (Sigma-Aldrich, catalog number: I7125 )
5-Chloro-2’-deoxyuridine (CldU) (Sigma-Aldrich, catalog number: C6891 )
Chemicals of analytical grade

Methanol (CARLO ERBA Reagents, catalog number: 414814 )
Acetic acid glacial (Fisher Scientific, catalog number: A38-212 )
Ethanol absolute (CARLO ERBA Reagents, catalog number: 308605 )
39.5% hydrochloric acid (HCl) (CARLO ERBA Reagents, catalog number: 403878 )
Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A9418 )
Tween^® 20 (Sigma-Aldrich, catalog number: P9416 )
ProLong^® Gold anti-fade reagent (Thermo Fisher Scientific, Invitrogen^TM, catalog number: P36935 )
Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653 )
Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9541 )
Sodium phosphate dibasic (Na₂HPO₄) (Sigma-Aldrich, catalog number: 255793 )
Potassium phosphate monobasic (KH₂PO₄) (Sigma-Aldrich, catalog number: 795488 )
Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: 71725 )
Tris (Roche Diagnostics, catalog number: 10708976001 )
0.5 M EDTA (pH 8.0) (Thermo Fisher Scientific, Invitrogen^TM, catalog number: AM9262 )
NP-40 (Thermo Fisher Scientific, catalog number: 85124 )

Antibodies

Anti-BrdU clone BU1/75 (ICR1) (detects CldU) (Bio-Rad Laboratories, catalog number: OBT0030CX )
Anti-BrdU clone B44 (detects IdU) (BD, BD Biosciences, catalog number: 347580 )
Alexa Fluor 488-conjugated chicken anti-rat (Thermo Fisher Scientific, Invitrogen, catalog number: A21470 )
Alexa Fluor 594-conjugated rabbit anti-mouse (Thermo Fisher Scientific, Invitrogen, catalog number: A-11062 )

1x phosphate buffered saline (PBS) (see Recipes)
Spreading buffer (see Recipes)
Stringent buffer (see Recipes)

Equipment

Incubator
UVC 500 UV Crosslinker (GE Healthcare, model: UVC 500 )
Centrifuge
Confocal microscope (e.g., Carl Zeiss, model: Zeiss LSM100 )
Fume hood

Software

GraphPad software

Procedure

English

中文翻译

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如何引用

Luigi, A., Giordano, A. and Pentimalli, F. (2017). DNA Fiber Assay upon Treatment with Ultraviolet Radiations. Bio-protocol 7(11): e2301. DOI: 10.21769/BioProtoc.2301.