Name: laser microirradiation to study in vivo cellular responses to simple and complex dna damage
Uploaded: 2018-01-31T12:30:00
Description: Watch this Scientific Journal Video about Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage at JoVE.com

We thank Dr. Akira Yasui at Tohoku University, Japan for the GFP-NTH1 expression plasmid, and Dr. Eros Lazzerini Denchi at the Scripps Research Institute, La Jolla, California for the TRF2-YFP and EGFP-53BP11220-1711 expression plasmids. This work was supported by the Air Force Office of Scientific Research (FA9550-04-1-0101) and the Beckman Laser Institute Inc. Foundation (to M.W.B), the Ford Foundation Fellowship from the National Academy of Sciences (to B.A.S), and NSF MCB-1615701 and CRCC CRR-17-426665 (to K. Y.).

1. Basic Cell Preparation
Note: This step is for standard immunofluorescence detection of the endogenous protein recruitment or modification, and for the use of a cell line stably expressing a fluorescently tagged recombinant protein. For example, Potorustridactylus (PtK2) marsupial kidney epithelial cells stably expressing EGFP-53BP11220-1711 or TRF2-YFP were used in our previous study (Figure 1)32. In the former case, th...

The advantages of using laser microirradiation for DDR research are:
1. It is feasible to induce different types and amounts of DNA damage from simple strand breaks to complex DNA damage, and to detect different repair factors at the DNA damage sites by adjusting the laser irradiation parameters. It is also possible to inflict damage multiple times in the same cell nucleus in order to evaluate trans effects (as in Figure 3).
2....

In Vivo DNA Damage Signaling is Not Well Understood 
In vivo, DNA is complexed with histones and other factors to form chromatin fibers. Regulation of chromatin structure is of paramount importance for DNA metabolism. For example, the histone variant H2AX is phosphorylated by ataxia-telangiectasia mutated (ATM) and other kinases following double-strand breaks (DSB) induction, and is important for DSB damage signal amplification as well as providing a docking site for other factors. Spreading of damage signaling and repair pathway choice appear to be critically influenced by local chromatin structure at damage sites1. A number of chromatin remodeling factors, histone chaperones, and histone modifying enzymes are indeed recruited to damage sites and are important for efficient DNA repair, highlighting the significance of chromatin regulation in the DDR and repair2,3,4. Furthermore, damage site clustering or repositioning was observed in yeast and drosophila5,6,7,8, reminiscent of relocalization of gene loci in the subnuclear compartment associated with gene regulation9,10. Recent studies in mouse and human cells also revealed mobilization of DSB sites, which influences repair fidelity and pathway choice11,12. This raises the possibility that DDR/repair may also be intimately linked to nuclear architecture, higher-order chromatin organization, and chromosome dynamics in the cell nucleus. Thus, it is critical to develop high-resolution methods to study DDR and repair processes in the context of the endogenous nuclear environment in a live cell in order to understand short- and long-term consequences of DNA damage.
Critical Role of PAR Polymerase (PARP) in Gauging the Extent and Type of Damage and Regulating Protein Assembly at the Damage Site 
PARP1 is a DNA nick sensor rapidly activated by DNA damage that plays a critical role in DNA repair13. PARP1 was originally thought to function together with X-Ray Repair Cross Complementing 1 (XRCC1) to facilitate base excision repair (BER), but recent studies reveal its role in other DNA repair pathways, including DSB repair14. Activated PARP1 uses nicotinamide adenine dinucleotide (NAD+) as a substrate to ADP-ribosylate multiple target proteins, including itself. This enzyme and other family members have attracted much attention in recent years as PARP inhibitors have emerged as promising therapeutic drugs for cancers. Although PARP inhibitors were initially found to be effective in breast cancer gene (BRCA)-mutated breast cancer cells, there is now a plethora of evidence for their effects in mono- and combination therapies together with DNA damaging agents/irradiation against a broad spectrum of cancers with mutations not limited to BRCA15,16,17,18,19,20.
At the molecular level, PARP activation was shown to play critical roles in organizing the local chromatin structure at damage sites. PAR-dependent recruitment of chromatin modification enzymes facilitates DSB repair and dictates repair pathway choices, suggesting the important scaffolding role of PAR modification at damage sites. 13,21,22,23,24,25,26,27,28,29,30,31 We recently demonstrated the exclusion of p53-binding protein 1 (53BP1) from damage sites by PAR 32, providing an alternative explanation for 53BP1-dependent hyperactivation of non-homologous endjoining (NHEJ) by PARP inhibitor and highlighting the significance of PARP in DSB repair pathway choice33,34. PARP1 also directly PARylates and affects the activity of multiple DNA repair factors14.
Using Laser Microirradiation as a Tool to Study DDR/Repair In Vivo 
Laser microirradiation to produce sub-micron alterations on individual chromosomes was first described in 196935 and reviewed in detail in 198136. Several decades later, laser microirradiation was shown to induce DNA damage at a defined submicrometer region in the cell nucleus, and was proven to be a valuable technique to study recruitment or modifications of various factors to DNA lesions in vivo13,37,38,39,40,41. This method allows detection of those factors that do not form distinct irradiation-induced foci (IRIF) at damage sites39,42. It is also possible to examine the spatiotemporal dynamics of chromatin structural changes both at damage sites and in the rest of the nucleus. We carefully compared the DDRs induced by different laser systems and input powers to evaluate the relationship between the type of DNA damage and the microirradiation conditions32,43,44,45. The aberrant recruitment patterns of 53BP1 and telomeric repeat binding factor 2 (TRF2) were observed in the previous laser damage studies, which provided the basis for the recurring concern for the &#34;non-physiological&#34; nature of laser-induced damage46,47,48,49. We found that these apparent discrepancies could now be explained by differential PARP signaling which gauges the amount and complexity of induced damage32. We confirmed that: 1) laser-microirradiated cells (even after high input-power irradiation) are arrested in interphase in a damage checkpoint control-dependent manner and remain viable (at least up to 48 h)32,50; and 2) repair factor recruitment/modifications faithfully recapitulate those observed with treatment with conventional DNA damaging agents and DSB induction by endonucleases32,39,42,44,50,51,52. These results strongly support the physiological relevance of studying laser damage-induced cellular responses.

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laser microirradiation to study in vivo cellular responses to simple and complex dna damage

DNA damage induces specific signaling and repair responses in the cell, which is critical for protection of genome integrity. Laser microirradiation became a valuable experimental tool to investigate the DNA damage response (DDR) in vivo. It allows real-time high-resolution single-cell analysis of macromolecular dynamics in response to laser-induced damage confined to a submicrometer region in the cell nucleus. However, various laser conditions have been used without appreciation of differences in the types of damage induced. As a result, the nature of the damage is often not well characterized or controlled, causing apparent inconsistencies in the recruitment or modification profiles. We demonstrated that different irradiation conditions (i.e., different wavelengths as well as different input powers (irradiances) of a femtosecond (fs) near-infrared (NIR) laser) induced distinct DDR and repair protein assemblies. This reflects the type of DNA damage produced. This protocol describes how titration of laser input power allows induction of different amounts and complexities of DNA damage, which can easily be monitored by detection of base and crosslinking damages, differential poly (ADP-ribose) (PAR) signaling, and pathway-specific repair factor assemblies at damage sites. Once the damage conditions are determined, it is possible to investigate the effects of different damage complexity and differential damage signaling as well as depletion of upstream factor(s) on any factor of interest.

Using PtK2 cells stably expressing EGFP-53BP11220-1711 or TRF2-YFP, laser input power titration was carried out to determine the optimal condition for their recruitment (Figure 1)32.
At high input-power laser damage sites, the significant clustering of CPD (crosslinking damage typically generated by ultraviolet (UV) damage) as well as GFP-NTH1 DNA glycosylase that specifically recognizes base damage were observed. In addition, hi...

Watch this Scientific Journal Video about Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage at JoVE.com

Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage

The goal of this protocol is to describe how to use laser microirradiation to induce different types of DNA damage, including relatively simple strand breaks and complex damage, to study DNA damage signaling and repair factor assembly at damage sites in vivo.

Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage

DNA damage induces specific signaling and repair responses in the cell, which is critical for protection of genome integrity. Laser microirradiation ...

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JoVE Journal

Genetics

Genetic Manipulation of the Plant Pathogen Ustilago maydis to Study Fungal Biology and Plant Microbe Interactions

Genetic Manipulation of the Plant Pathogen Ustilago maydis to Study Fungal Biology and Plant Microbe Interactions

Gene deletion plays an important role in the analysis of gene function. One of the most efficient methods to disrupt genes in a targeted manner is the ...

Tools to Study the Role of Architectural Protein HMGB1 in the Processing of Helix Distorting, Site-specific DNA Interstrand Crosslinks

High mobility group box 1 (HMGB1) protein is a non-histone architectural protein that is involved in regulating many important functions in the genome, ...

Peptide-derived Method to Transport Genes and Proteins Across Cellular and Organellar Barriers in Plants

The capacity to introduce exogenous proteins and express (or down-regulate) specific genes in plants provides a powerful tool for fundamental research as ...

Analysis of Cap-binding Proteins in Human Cells Exposed to Physiological Oxygen Conditions

Translational control is a focal point of gene regulation, especially during periods of cellular stress. Cap-dependent translation via the eIF4F complex ...

Chromatin Immunoprecipitation (ChIP) Protocol for Low-abundance Embryonic Samples

Chromatin Immunoprecipitation ChIP Protocol for Low-abundance Embryonic Samples

Chromatin immunoprecipitation (ChIP) is a widely-used technique for mapping the localization of post-translationally modified histones, histone variants, ...

The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions

The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions

We demonstrate a method using Caenorhabditis elegans as a model host to study microbial interaction. Microbes are introduced via the diet making the ...

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA ...

Simultaneous Video-EEG-ECG Monitoring to Identify Neurocardiac Dysfunction in Mouse Models of Epilepsy

In epilepsy, seizures can evoke cardiac rhythm disturbances such as heart rate changes, conduction blocks, asystoles, and arrhythmias, which can ...

Investigation of Protein Recruitment to DNA Lesions Using 405 Nm Laser Micro-irradiation

The DNA Damage Response (DDR) uses a plethora of proteins to detect, signal, and repair DNA lesions. Delineating this response is critical to understand ...

A Simple, Rapid, and Quantitative Assay to Measure Repair of DNA-protein Crosslinks on Plasmids Transfected into Mammalian Cells

The purpose of this method is to provide a flexible, rapid, and quantitative technique to examine the kinetics of DNA-protein crosslink (DPC) repair in ...