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.).

The authors have nothing to disclose.

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. Importantly, events happening at damage sites and the secondary events that occur elsewhere in the nucleus can easily be distinguished.
3. It is possible to carry out high-resolution real-time measurements of fluorescently tagged protein dynamics in response to damage at a single cell level, including, but not limited to: F&#246;rster Resonance Energy Transfer (FRET), Fluorescence Lifetime Imaging (FLIM), Pair Correlation Function (pCF), etc., to capture DDR in live cells.
4 .Combining with RNA interference or inhibitor treatment, it is relatively straightforward to test upstream factor requirement in a small number of cells.
5. It should also be noted that NIR (or green) laser radiation does not require pre-sensitization of DNA with nucleotide analogs or DNA-intercalating dyes, thus avoiding unwanted side effects on chromatin packing. This is in contrast to the UV laser system that requires sensitization at low dose and causes aberrant DDR at high dose39,49,61.
Critical Steps Within the Protocol/Modifications and Troubleshooting 
It is important that the cells are in healthy conditions when DDR analyses are performed in order to minimize artifact and experimental variations. DNA- or siRNA-transfected cells usually are more sensitive or easily detached when growing on the gridded coverslip. It helps to keep cells for shorter times in the transfection reagent as long as the transfection works, and seed more transfected cells onto the gridded coverslip dish compared to the untransfected cells. This is necessary in order to achieve comparable cell confluences for the experiments.
It has been reported that the thymidine block leads to increased levels of &#947;H2AX in the synchronized cells62, which may affect DDR and skew the results. Furthermore, the base line &#947;H2AX signal was shown to be higher in S and G2/M phase even without any exogenous damage63,64. We, however, do not observe any significant &#947;H2AX signal in the nucleoplasm that would affect or interfere with the specific &#947;H2AX response at laser-induced damage sites in the cells synchronized in S/G2 phase by a double thymidine block51.
In order to obtain consistent results, it is necessary to periodically check the laser system output parameters (every 2 to 4 weeks) and optical alignment (every 2 months). Over time, laser system components may need to be re-aligned, objective lens may be damaged and need to be replaced, and the stability of the total input power may change. The key is to evaluate the presence of damage (crosslinking (CPD and/or 6-4PP) and base damage (8-oxoG)), marker proteins (e.g., DNA glycosylases, Rad51, cohesin, and Ku), and PAR modification described in this protocol to determine the dosage and complexity of DNA damage induced by the laser microscope system used.
Limitations of the Technique 
Laser microirradiation has limitations. DNA damage induced by the laser beam is highly clustered in one area in the nucleus compared to naturally occurring damage that may be spread throughout the nucleus. This may change how damage signaling is propagated in the neighboring chromatin or in the nucleus. Since a relatively low number of cells can be irradiated at one time, population-based biochemical assays (e.g., Western blot, protein complex purification, chromatin immunoprecipitation (ChIP)) cannot be easily performed. Reliance on fluorescently tagged proteins (with over expression of the exogenous recombinant proteins or even with those expressed endogenously using CRISPR-mediated tag insertion) for dynamic imaging makes the studies vulnerable to protein fusion-induced artifacts. The unique nature of laser-induced damage and potential artifactual effects of tagged proteins, therefore, need to be evaluated by comparison with the results using conventional DNA damaging methods (IR, chemical damaging agents) and with the endogenous untagged proteins (though sometimes it is not possible to perform complete parallel experiments).
Significance with Respect to Existing Methods 
The use of sequence-specific endonucleases such as I-SceI, FokI, AsiSI, and I-PpoI provides an alternative strategy to induce site-specific DSBs (strictly simple DSBs). Factor recruitment or modifications can be analyzed by site-specific or genome-wide ChIP analysis65,66,67,68,69. Relatively synchronous induction of DSBs can be achieved by tagging the endonuclease with a steroid hormone receptor and a degradation signal (degron) for rapid nuclear translocation and degradation, respectively65,66,67,68,69. One must take into consideration, however, that the efficiency of endonuclease expression and target site accessibility, and ongoing repair status can be variable in different cells and at different target sites. These heterogeneities will be averaged in the population-based ChIP analysis, which may skew the results. Laser microirradiation, on the other hand, offers the highest possible temporal resolution (milliseconds) as well as spatial resolution (submicrometer) of damage response dynamics, which can be analyzed at the single-cell level. High damage density, however, may influence the outcome. Both strategies can be sensitive to artifacts caused by antibody accessibility and/or peptide tagging. It is, therefore, important to understand the pros and cons of both strategies, which may potentially be used to complement each other.
Future Applications 
With rapid advancement of fluorescence imaging and dynamics techniques, versatility of laser systems to induce different amounts and types of DNA damage at specific locations and cell cycle stage provides a valuable opportunity to interrogate the cellular and molecular responses to DNA damage with high spatiotemporal resolution at a single cell level. It will be possible, for example, to tease out damage site-specific and nucleus- and cell-wide DDR signal transduction with millisecond-resolution (e.g., detect molecular interactions or modifications that occur exclusively at damage sites, examine dynamic changes of chromatin structure in real-time, or observe real-time spreading of damage signaling from damage sites to the whole nucleus). It is also possible to follow the same cell over a long period of time to examine the effects of DNA damage on cell fate. We envision that the laser microirradiation methods, if used properly, will continue to contribute significantly to advancement of the DNA repair field.

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.

<table border="0" cellpadding="0" cellspacing="0" width="1172">
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			<td height="21" style="height:21px;width:216px;">HeLa cells</td>
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			<td>supplemented with L-Glutamine, 4% FBS</td>
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			<td style="width:251px;">generated in KY lab</td>
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			<td style="width:441px;">ref 43</td>
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		<tr height="42">
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			<td style="width:251px;">generated in KY lab</td>
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			<td style="width:251px;">generated in KY lab</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">GFP-NEIL2 stable cell line</td>
			<td style="width:251px;">generated in KY lab</td>
			<td style="width:264px;"></td>
			<td style="width:441px;">ref 54</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">GFP-NTH1 stable cell line</td>
			<td style="width:251px;">generated in KY lab</td>
			<td style="width:264px;"></td>
			<td style="width:441px;">ref 32</td>
		</tr>
		<tr height="21">
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			<td style="width:251px;">generated in KY lab</td>
			<td style="width:264px;"></td>
			<td style="width:441px;"></td>
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			<td style="width:251px;">generated in KY lab</td>
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			<td style="width:251px;">generated in Berns lab</td>
			<td style="width:264px;"></td>
			<td style="width:441px;">ref 32</td>
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		<tr height="21">
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			<td style="width:251px;">generated in Berns lab</td>
			<td style="width:264px;"></td>
			<td>ref 32</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Olaparib&#160;</td>
			<td style="width:251px;">Apexbio Technology</td>
			<td>A4154</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Paraformaldehyde&#160;</td>
			<td>ELECTRON MICROSCOPY SCIENC MS</td>
			<td style="width:264px;">100503-916 (EA)</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Quantity One 1-D Analysis Software Version 4.6.9</td>
			<td style="width:251px;">Bio-Rad</td>
			<td style="width:264px;">SOFT-LIT-70-9600-Q1-469PC&#160;</td>
			<td>image analysis program</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Excel</td>
			<td style="width:251px;">microsoft</td>
			<td style="width:264px;"></td>
			<td>spreadsheet program</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Triton X100</td>
			<td style="width:251px;">Fisher Scientific</td>
			<td style="width:264px;">BP151-500</td>
			<td>detergent</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Dulbecco&#39;s phosphate-buffered saline (DPBS) 10X without calcium and magnesium</td>
			<td style="width:251px;">Fisher Scientific</td>
			<td style="width:264px;">14200166</td>
			<td>dilute to 1X&#160;</td>
		</tr>
	</tbody>
</table>

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, higher XRCC1 and CtIP signals were observed, reflecting the increased number of strand breaks, and demonstrating that complex DNA damage is induced under this condition (Figure 2A). Other DNA glycosylases fused to fluorescent proteins (e.g., GFP-nei endonuclease VIII-like 2 (NEIL2) and GFP-8-Oxoguanine glycosylase (OGG1)) as well as condensin I can also be used as base damage markers44,59. Consistent with this, we found that the PAR response is induced significantly by high input-power laser (i.e., energy and irradiance in the focal spot in the specimen), but only weakly by low laser power in the focal spot (Figure 2B, top left). PAR signals, but not PARP1 protein localization, at damage sites were sensitive to the PARP inhibitor (data not shown). Additionally, siRNA specific for PARP1 diminished both PAR and PARP1 at damage sites (Figure 2B, top right)32. Under the high input power condition, &#947;H2AX initially appears at the damage sites, and rapidly spreads to the entire nucleus in an ATM/DNA-PK-dependent manner (Figure 2B, bottom). Similar ATM/DNA-PK-dependent spreading of &#947;H2AX was reported for &#947;-irradiation60. Consistent results were obtained with both 800 and 780 nm NIR laser systems32. Taken together, the results reveal that it is possible to titrate laser input power (and therefore, energy and irradiance in the focal spot in the cell) to find the conditions that induce different degrees of PARP response correlating with the number of DSBs and the complexity of the DNA damage.
The above results initially suggested that the presence of complex DNA damage might have caused differential recruitment of 53BP1 and TRF2. Interestingly, however, the 53BP11220-1711 recruitment to a low-power damage site was effectively inhibited by the presence of the second damage site induced by high-input-power in the same cell nucleus (Figure 3A in contrast to Figure 3B). The results indicate that high-input-power laser damage suppresses 53BP1 recruitment in trans. Inhibition of PAR by PARP inhibitor as well as inhibition of nuclear-wide &#947;H2AX spreading by ATM/DNA-PK inhibitors restore this recruitment even to the high input power damage site (Figure 3A). This is distinct from the recruitment of Mediator of DNA Damage Checkpoint 1 (MDC1) to damage sites, which was primarily inhibited by &#947;H2AX dispersion, and therefore, was restored by ATM/DNA-PK inhibitors, not PARP inhibitor (Figure 3A, bottom). Importantly, hyperactivation of PAR by PARG inhibition (without affecting &#947;H2AX status) effectively suppressed initial 53BP1 recruitment even at the low input-power damage sites that normally recruit 53BP1 efficiently (Figure 3B)32. Taken together, these results indicate that PAR signaling, and not the type of damage per se, interferes with the recruitment of 53BP132. This is an example of the versatility of laser microirradiation, which allows us to examine how multiple damage sites influence and interact with each other.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56213/56213fig1.jpg" /> 
Figure 1: Titration of laser input power. To determine the laser power to use for future experiments, PtK2 cells stably expressing EGFP-53BP1 and TRF2-YFP were subjected to laser microirradiation with input powers ranging between 20 mW and 155 mW using the 800 nm NIR laser/inverted epi-fluorescence microscope system. EGFP-53BP1 recruitment was monitored for 15 min post irradiation (p.i.), while TRF2-YFP cells were followed for 6 min p.i. The numbers directly above the bars represent the number of cells that showed positive recruitment of EGFP-53BP1 or TRF2-YFP to the damage sites over the total number of cells tested. This figure was modified from Saquilabon Cruz32. <a href="/files/ftp_upload/56213/56213fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56213/56213fig2.jpg" /> 
Figure 2: Induction of complex damage and robust DDR signaling by high input-power laser. (A) High-input-power laser microirradiation induces complex damage including strand breaks (both SSBs and DSBs), crosslinking, and base damage. CPD, XRCC1 (SSB and DSB repair), NTH1 DNA glycosylase (BER), and CtIP (alternative NHEJ and HR) are shown (only GFP-NTH1 is a live cell image and the rest are observed following immunofluorescence staining). Quantitative fluorescence intensity measurements of the damage site recruitment were done as stated in protocol Section 7 and are shown in the boxplots. The total number of cells (n) tested can be seen under each quantified protein. *p-value &#60;0.05. (B) Top (left): Robust PAR and PARP1 signals at high input-power damage sites. Top (right): PARP1 siRNA depletion is sufficient to abolish the PAR signal32,44. Bottom: Time course analysis of &#947;H2AX in cells with low (top) and high (bottom) input power damage as indicated. Scale bars = 10 &#181;m. This figure was modified from Saquilabon Cruz32. <a href="/files/ftp_upload/56213/56213fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56213/56213fig3.jpg" /> 
Figure 3: Differential activation of DDR signaling affects 53BP1 recruitment to damage sites. (A) Top: PtK2 cells stably expressing EGFP-53BP11220-1711 were microirradiated with both low (15%) and high (25%) input power (white and yellow arrowheads, respectively) in the same nucleus using the 780 nm NIR/confocal microscope system. This was performed in the presence of DMSO, PARP inhibitor (Pi), or the combination of ATM, DNA-PK, and PARP inhibitors (Ai+Di+Pi) as indicated. Live-cell imaging of EGFP-53BP11220-1711 was captured at 30 min after DSB induction. Cells were then fixed and stained with an antibody specific for &#947;H2AX as a DSB marker. Changes of fluorescence intensity of EGFP-53BP11220-1711 at damage sites are shown on the right with p-values (mean values are: DMSO, 0.03 &#177; 0.02; Pi, 0.34 &#177; 0.19; Ai+Di+Pi, 0.98 &#177; 0.23). Bottom: The effect of Pi and Ai+Di treatment on MDC1 localization with high input-power damage as indicated. Scale bar is 10 &#181;m. Right: Changes of fluorescence signals of MDC1 at high input-power damage sites in the presence of DMSO, Pi, or Ai+Di with p-values (mean values are: DMSO, 0.07 &#177; 0.10; Pi, 0.05 &#177; 0.07; Ai+Di, 0.86 &#177; 0.26). Scale bar = 10 &#181;m. N = 10 for each intensity measurement. (B) The effect of enhancement of PAR signals by PARGi treatment on the endogenous 53BP1 recruitment to low input-power damage sites. Right: Boxplots representing the quantitative analysis of endogenous 53BP1 recruitment (detected by immunofluorescence staining) with low input-power laser (60 mW in the 800 nm NIR laser/inverted epi-fluorescence microscope system) at 15 min post irradiation in the presence of DMSO or PARGi as indicated. Left: PAR and 53BP1 immunofluorescence staining pictures of cells damaged under the same conditions with DMSO or PARGi treatment. For DMSO treatment, 100% of cells examined (N = 25) exhibited robust 53BP1 recruitment (right). In contrast, 20/25 showed no 53BP1 recruitment in the presence of PARGi (bottom right), and 5 cells showed weak recruitment. Scale bar = 10 &#181;m. This figure was modified from Saquilabon Cruz32. <a href="/files/ftp_upload/56213/56213fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

laser-microirradiation-to-study-vivo-cellular-responses-to-simple

Nanyang Technological University

Research

JoVE Journal

Genetics

10.1K Views.  University of California, Irvine. 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.

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

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 replacement of the entire gene with a selectable marker via homologous recombination. During homologous recombination, exchange of DNA takes place between sequences with high similarity. Therefore, linear genomic sequences flanking a target gene can be used to specifically direct a selectable marker to the desired integration site. Blunt ends of the deletion construct activate the cell&#39;s DNA repair systems and thereby promote integration of the construct either via homologous recombination or by non-homologous-end-joining. In organisms with efficient homologous recombination, the rate of successful gene deletion can reach more than 50% making this strategy a valuable gene disruption system. The smut fungus Ustilago maydis is a eukaryotic model microorganism showing such efficient homologous recombination. Out of its about 6,900 genes, many have been functionally characterized with the help of deletion mutants, and repeated failure of gene replacement attempts points at essential function of the gene. Subsequent characterization of the gene function by tagging with fluorescent markers or mutations of predicted domains also relies on DNA exchange via homologous recombination. Here, we present the U. maydis strain generation strategy in detail using the simplest example, the gene deletion.

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

We describe a robust gene replacement strategy to genetically manipulate the smut fungus Ustilago maydis. This protocol explains how to generate deletion mutants to investigate infection phenotypes. It can be extended to modify genes in any desired way, e.g., by adding a sequence encoding a fluorescent protein tag.

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, such as transcription, DNA replication, and DNA repair. HMGB1 binds to structurally distorted DNA with higher affinity than to canonical B-DNA. For example, we found that HMGB1 binds to DNA interstrand crosslinks (ICLs), which covalently link the two strands of the DNA, cause distortion of the helix, and if left unrepaired can cause cell death. Due to their cytotoxic potential, several ICL-inducing agents are currently used as chemotherapeutic agents in the clinic. While ICL-forming agents show preferences for certain base sequences (e.g., 5&#39;-TA-3&#39; is the preferred crosslinking site for psoralen), they largely induce DNA damage in an indiscriminate fashion. However, by covalently coupling the ICL-inducing agent to a triplex-forming oligonucleotide (TFO), which binds to DNA in a sequence-specific manner, targeted DNA damage can be achieved. Here, we use a TFO covalently conjugated on the 5&#39; end to a 4&#39;-hydroxymethyl-4,5&#39;,8-trimethylpsoralen (HMT) psoralen to generate a site-specific ICL on a mutation-reporter plasmid to use as a tool to study the architectural modification, processing, and repair of complex DNA lesions by HMGB1 in human cells. We describe experimental techniques to prepare TFO-directed ICLs on reporter plasmids, and to interrogate the association of HMGB1 with the TFO-directed ICLs in a cellular context using chromatin immunoprecipitation assays. In addition, we describe DNA supercoiling assays to assess specific architectural modification of the damaged DNA by measuring the amount of superhelical turns introduced on the psoralen-crosslinked plasmid by HMGB1. These techniques can be used to study the roles of other proteins involved in the processing and repair of TFO-directed ICLs or other targeted DNA damage in any cell line of interest.

Targeted DNA damage can be achieved by tethering a DNA damaging agent to a triplex-forming oligonucleotide (TFO). Using modified TFOs, DNA damage-specific protein association, and DNA topology modification can be studied in human cells by the utilization of modified chromatin immunoprecipitation assays and DNA supercoiling assays described herein.

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 well as new applications in the field of plant biotechnology. Viable methods that currently exist for protein or gene transfer into plant cells, namely Agrobacterium and microprojectile bombardment, have disadvantages of low transformation frequency, limited host range, or a high cost of equipment and microcarriers. The following protocol outlines a simple and versatile method, which employs rationally-designed peptides as delivery agents for a variety of nucleic acid- and protein-based cargoes into plants. Peptides are selected as tools for development of the system due to their biodegradability, reduced size, diverse and tunable properties as well as the ability to gain intracellular/organellar access. The preparation, characterization and application of optimized formulations for each type of the wide range of delivered cargoes (plasmid DNA, double-stranded DNA or RNA, and protein) are described. Critical steps within the protocol, possible modifications and existing limitations of the method are also discussed.

Existing methods for the modification of plants have limited applicability. The novel peptide-derived technology proposed here promises both simplicity and efficiency in the introduction of exogenous protein or genes into the desired intracellular compartments of intact plants.

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

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, transcription factors, or chromatin-modifying enzymes at a given locus or on a genome-wide scale. The combination of ChIP assays with next-generation sequencing (i.e., ChIP-Seq) is a powerful approach to globally uncover gene regulatory networks and to improve the functional annotation of genomes, especially of non-coding regulatory sequences. ChIP protocols normally require large amounts of cellular material, thus precluding the applicability of this method to investigating rare cell types or small tissue biopsies. In order to make the ChIP assay compatible with the amount of biological material that can typically be obtained in vivo during early vertebrate embryogenesis, we describe here a simplified ChIP protocol in which the number of steps required to complete the assay were reduced to minimize sample loss. This ChIP protocol has been successfully used to investigate different histone modifications in various embryonic chicken and adult mouse tissues using low to medium cell numbers (5 x 104 - 5 x 105 cells). Importantly, this protocol is compatible with ChIP-seq technology using standard library preparation methods, thus providing global epigenomic maps in highly relevant embryonic tissues.

Chromatin Immunoprecipitation ChIP Protocol for Low-abundance Embryonic Samples

Here, we describe a chromatin immunoprecipitation (ChIP) and ChIP-seq library preparation protocol to generate global epigenomic profiles from low-abundance chicken 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

We demonstrate a method using Caenorhabditis elegans as a model host to study microbial interaction. Microbes are introduced via the diet making the intestine the primary location for disease. The nematode intestine structurally and functionally mimics mammalian intestines and is transparent making it amenable to microscopic study of colonization. Here we show that pathogens can cause disease and death. We are able to identify microbial mutants that show altered virulence. Its conserved innate response to biotic stresses makes C. elegans an excellent system to probe facets of host innate immune interactions. We show that hosts with mutations in the dual oxidase gene cannot produce reactive oxygen species and are unable to resist microbial insult. We further demonstrate the versatility of the presented survival assay by showing that it can be used to study the effects of inhibitors of microbial growth. This assay may also be used to discover fungal virulence factors as targets for the development of novel antifungal agents, as well as provide an opportunity to further uncover host-microbe interactions. The design of this assay lends itself well to high throughput whole-genome screens, while the ability to cryo-preserve worms for future use makes it a cost-effective and attractive whole animal model to study.

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

Here, we present the nematode Caenorhabditis elegans as a versatile host model to study microbial interaction.

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

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA mutation rates. Changes in replication timing occur during development and in cancer, but the role replication timing plays in development and disease is not known. Zebrafish were recently established as an in vivo model system to study replication timing. Here is detailed the protocols for using the zebrafish to determine DNA replication timing. After sorting cells from embryos and adult zebrafish, high-resolution genome-wide DNA replication timing patterns can be constructed by determining changes in DNA copy number through analysis of next generation sequencing data. The zebrafish model system allows for evaluation of the replication timing changes that occur in vivo throughout development, and can also be used to assess changes in individual cell types, disease models, or mutant lines. These methods will enable studies investigating the mechanisms and determinants of replication timing establishment and maintenance during development, the role replication timing plays in mutations and tumorigenesis, and the effects of perturbing replication timing on development and disease.

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

Zebrafish were recently used as an in vivo model system to study DNA replication timing during development. Here is detailed the protocols for using zebrafish embryos to profile replication timing. This protocol can be easily adapted to study replication timing in mutants, individual cell types, disease models, and other species.

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 potentially increase risk of sudden unexpected death in epilepsy (SUDEP). Electroencephalography (EEG) and electrocardiography (ECG) are widely used clinical diagnostic tools to monitor for abnormal brain and cardiac rhythms in patients. Here, a technique to simultaneously record video, EEG, and ECG in mice to measure behavior, brain, and cardiac activities, respectively, is described. The technique described herein utilizes a tethered (i.e., wired) recording configuration in which the implanted electrode on the head of the mouse is hard-wired to the recording equipment. Compared to wireless telemetry recording systems, the tethered arrangement possesses several technical advantages such as a greater possible number of channels for recording EEG or other biopotentials; lower electrode costs; and greater frequency bandwidth (i.e., sampling rate) of recordings. The basics of this technique can also be easily modified to accommodate recording other biosignals, such as electromyography (EMG) or plethysmography for assessment of muscle and respiratory activity, respectively. In addition to describing how to perform the EEG-ECG recordings, we also detail methods to quantify the resulting data for seizures, EEG spectral power, cardiac function, and heart rate variability, which we demonstrate in an example experiment using a mouse with epilepsy due to Kcna1 gene deletion. Video-EEG-ECG monitoring in mouse models of epilepsy or other neurological disease provides a powerful tool to identify dysfunction at the level of the brain, heart, or brain-heart interactions.

Here, we present a protocol to record brain and heart bio signals in mice using simultaneous video, electroencephalography (EEG), and electrocardiography (ECG). We also describe methods to analyze the resulting EEG-ECG recordings for seizures, EEG spectral power, cardiac function, and heart rate variability.

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 genome maintenance mechanisms. Since recruitment and exchange of proteins at lesions are highly dynamic, their study requires the ability to generate DNA damage in a rapid and spatially-delimited manner. Here, we describe procedures to locally induce DNA damage in human cells using a commonly available laser-scanning confocal microscope equipped with a 405 nm laser line. Accumulation of genome maintenance factors at laser stripes can be assessed by immunofluorescence (IF) or in real-time using proteins tagged with fluorescent reporters. Using phosphorylated histone H2A.X (&#947;-H2A.X) and Replication Protein A (RPA) as markers, the method provides sufficient resolution to discriminate locally-recruited factors from those that spread on adjacent chromatin. We further provide ImageJ-based scripts to efficiently monitor the kinetics of protein relocalization at DNA damage sites. These refinements greatly simplify the study of the DDR dynamics.

Studying DNA damage repair kinetics requires a system to induce lesions at defined sub-nuclear regions. We describe a method to create localized double-stranded breaks using a laser-scanning confocal microscope equipped with a 405 nm laser and provide automated procedures to quantify the dynamics of repair factors at these lesions.

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 mammalian cell lines. Rather than globally assaying removal of xenobiotic-induced or spontaneous chromosomal DPC removal, this assay examines the repair of a homogeneous, chemically defined lesion specifically introduced at one site within a plasmid DNA substrate. Importantly, this approach avoids the use of radioactive materials and is not dependent on expensive or highly-specialized technology. Instead, it relies on standard recombinant DNA procedures and widely available real-time, quantitative polymerase chain reaction (qPCR) instrumentation. Given the inherent flexibility of the strategy utilized, the size of the crosslinked protein, as well as the nature of the chemical linkage and the precise DNA sequence context of the attachment site can be varied to address the respective contributions of these parameters to the overall efficiency of DPC repair. Using this method, plasmids containing a site-specific DPC were transfected into cells and low molecular weight DNA recovered at various times post-transfection. Recovered DNA is then subjected to strand-specific primer extension (SSPE) using a primer complementary to the damaged strand of the plasmid. Since the DPC lesion blocks Taq DNA polymerase, the ratio of repaired to un-repaired DNA can be quantitatively assessed using qPCR. Cycle threshold (CT) values are used to calculate percent repair at various time points in the respective cell lines. This SSPE-qPCR method can also be used to quantitatively assess the repair kinetics of any DNA adduct that blocks Taq polymerase.

The goal of this protocol is to quantify the repair of defined DNA-protein crosslinks on plasmid DNA. Lesioned plasmids are transfected into recipient mammalian cell lines and low-molecular weight harvested at multiple time points post-transfection. DNA repair kinetics are quantified using strand-specific primer extension followed by qPCR.

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 ...

Biotin-based Pulldown Assay to Validate mRNA Targets of Cellular miRNAs

MicroRNAs (miRNAs) are a class of small noncoding RNAs that post-transcriptionally regulate cellular gene expression. MiRNAs bind to the 3&#39; untranslated region (UTR) of target mRNA to inhibit protein translation or in some instances cause mRNA degradation. The binding of the miRNA to the 3&#39; UTR of the target mRNA is mediated by a 2&#8211;8 nucleotide seed sequence at the 5&#39; end of miRNA. While the role of miRNAs as cellular regulatory molecules is well established, identification of the target mRNAs with functional relevance remains a challenge. Bioinformatic tools have been employed to predict sequences within the 3&#39; UTR of mRNAs as potential targets for miRNA binding. These tools have also been utilized to determine the evolutionary conservation of such sequences among related species in an attempt to predict functional role. However, these computational methods often generate false positive results and are limited to predicting canonical interaction between miRNA and mRNA. Therefore, experimental procedures that measure direct binding of miRNA to its mRNA target are necessary to establish functional interaction. In this report, we describe a sensitive method for validating direct interaction between the cellular miRNA miR-125b and the 3&#39; UTR of PARP-1 mRNA. We elaborate a protocol in which synthetic biotinylated-miRNA mimics were transfected into mammalian cells and the miRNA-mRNA complex in the cellular lysate was pulled down with streptavidin-coated magnetic beads. Finally, the target mRNA in the pulled-down nucleic acid complex was quantified using a qPCR-based strategy.

This report describes a fast and reliable method for validating mRNA targets of cellular miRNAs. The method uses synthetic biotinylated Locked Nucleic Acid (LNA)-based miRNA mimics to capture target mRNA. Subsequently, streptavidin-coated magnetic beads are employed to pulldown the target mRNA for quantification by qPCR polymerase chain reaction.