Name: laser microirradiation to study in vivo cellular responses to simple and complex dna damage
Uploaded: 2018-01-31T12:30:00.000+00:00
Duration: 10 min 44 s
Description: Watch this Scientific Journal Video about Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage at JoVE.com

我们感谢 Dr. 野在东北大学, 日本 GFP-NTH1 表达质粒, 和 Dr. 爱神 Lazzerini Denchi 在斯研究所, la, 加利福尼亚为 TRF2-YFP 和 EGFP-53BP11220-1711表达的粒。这项工作得到了空军科研办公室 (FA9550-04-1-0101) 和贝克曼激光研究所 Inc. 基金会 (m.w. B)、美国国家科学院福特基金会奖学金 (美国)、NSF MCB-1615701 和中国CRR-17-426665 (阿莫亚科)。

1. 碱性细胞制备
注意: 这一步是为标准免疫荧光检测内源性蛋白质的招募或修改, 并为使用细胞系稳定表达荧光标记重组蛋白。例如, Potorustridactylus (PtK2) 袋肾上皮细胞稳定表达 EGFP-53BP11220-1711或 TRF2-YFP 在我们以前的研究 (图 1)32中使用。在前一种情况下, 53BP1 (氨基酸 1220-1711) 的焦点形成区, 其中包含的齐聚域, 都铎域, 和化依赖的招募 (UDR) 的主题, 被融合到增强 GFP (EGFP-53BP11220-1711)。这种融合蛋白重述的损伤现场招聘的全长 53BP153,54,55。在 HeLa 细胞中, 荧光标记的内聚力子单元 (例如、hSMC1-GFP51、GFP-Scc1 (Rad21)52和 GFP-SA2 51) 必须稳定地表达, 以确保有效地融入到复合体中, 并重述S/G2 细胞周期特定损伤部位的内源性内聚力的招募51。
<ol><li>
种子任何附着的细胞类型到一个35毫米的组织培养皿与格片, 以允许单独的细胞识别。根据特定细胞系的增殖速率, 调整初始细胞数以达到36-48 小时内的 60% 70-100。例如:
	<ol>
<li>为 PtK2 袋肾上皮细胞 (狂放的类型或那些稳定地表达 EGFP-53BP11220-1711或 TRF2-YFP): 在 2 mL 高级最低必需培养基 (记忆) 中板 2.0 x 104细胞补充2毫米 l-谷氨酰胺, 4% 胎牛血清 (FBS) 和抗生素 (100 U/毫升青霉素和100µg/毫升链霉素), 并孵育在37° c 与 7% CO2。</li>
		<li>对于稳定表达荧光标记重组蛋白的 HeLa 细胞 GFP-SA2: 种子 1.0 x 105细胞在2毫升 Dulbecco 的改良鹰的培养基 (DMEM) 补充 10% FBS, 2 毫米 l-谷氨酰胺, 100 U/毫升青霉素, 100 µg/毫升链霉素,和孵育在37° c 与 5% CO2。其他 HeLa 细胞也可用于包括未修饰的细胞或表达 hSMC1-GFP 的细胞。</li>
	</ol>
</li>
	<li>允许细胞坚持和增殖36-48 小时前 DNA 损伤诱导在30-60% 汇合 (允许可视化网格数)。进入4条。</li>
</ol>2. 瞬态转染
注意: 有时, 好的抗体不能用于检测内源性蛋白质。如果不具备稳定表达标记蛋白的细胞系, 可进行瞬态染监测荧光标记蛋白的活细胞分析。
<ol><li>对于 HeLa 细胞, 在1天, 种子 6-8 x 104细胞在0.5 毫升培养基中的 24-井板中, 在100% ° c 和 37 CO5%中孵育, 并在一只很好的2个平板中孵化。有关区域性媒体条件, 请参见步骤1.1。</li>
	<li>2天, 使用哺乳动物表达质粒编码荧光标记的 DNA 修复因子 (例如, GFP-NTH1 或 GFP-OGG1 基损伤32,44,56, GFP-Ku 为高剂量 DSBs57, GFP-53BP132相对简单的 DSBs, 或其他感兴趣的蛋白质融合到荧光标签) 执行脂质体介导的 DNA 转染。在1.5 毫升管中稀释0.3 µg 质粒 DNA 到25µL 无血清培养培养基中。</li>
	<li>另外1.5 毫升管, 稀释1µL 脂质体的 DNA 转染试剂为25µL 无血清培养基, 并轻轻混合。</li>
	<li>将稀释的 dna 和转染剂轻轻混合, 在室温下孵育10分钟, 形成 dna 脂质复合物。</li>
	<li>用0.5 毫升培养基冲洗细胞一次, 取出介质, 并向单元格中添加0.5 毫升新鲜培养基 (如步骤 1.1)。</li>
	<li>在细胞中加入 DNA 脂质配合物。轻轻摇晃盘子几次。将单元格放回孵化器, 如步骤2.1 所示。</li>
	<li>4-6 小时后, 从细胞中取出培养基, 加入300µL 0.05% 胰蛋白酶-EDTA。去除它, 增加200µL 胰蛋白酶-EDTA, 并且孵育在37° c 孵化器 3-4 min。</li>
	<li>在确认细胞分离后, 加入800µL 培养基, 重细胞通过轻度移, 使细胞团簇破裂。将细胞悬浮液转移到一个格片的 35 mm 培养皿中, 并将培养基添加到2毫升的最后一卷。</li>
	<li>孵育孵化器中的细胞, 如步骤2.1 所示48小时, 然后进行4节。 注: 如果重组蛋白的表达是有毒的 (在2.8 步中孵化后转染细胞呈圆形或不规则形态), 缩短表达时间, 并在转染后16-20 小时执行激光 microirradiation (第4节), 只要蛋白质表达可以被证实 (为荧光检测, 参见步骤 4.3)。在这种情况下, 执行转染直接在35毫米的菜。种子 3.0 x 105 HeLa 细胞, 在35毫米的格片, 在 50-70% h. 染 DNA 质粒后达到约30汇合, 并在转染后改变培养基 2-6 h。如果蛋白质表达合理, 细胞出现健康, 则进行激光 microirradiation 12-20 小时。</li>
</ol>3. 细胞周期同步
注: 对于同源重组修复 (hr) 蛋白, 如 Rad51 和内聚力, 在 S/G2 阶段的细胞周期中, 招聘更为突出, 其中 hr 修复发生在51。因此, 要监测这些蛋白质的招募, 细胞同步到 S/G2 阶段是必要的。
<ol><li>
S/G2 相位同步
	<ol>
<li>使用双胸苷块协议进行单元同步, 以 S/G2 阶段50,51。在播种细胞在35毫米格片盘 (参见步 1.1) 之后, 孵育细胞与胸苷 (到最后的集中2.5 毫米) 在文化媒介 (和在步 1.1) 为 17 h 在37° c 和与 5% CO2。</li>
		<li>冲洗细胞与1毫升无胸苷培养基 (如在步骤 1.1) 两次, 并孵育细胞在2毫升无胸苷培养基 9 h, 在步骤1.1。</li>
		<li>添加200µL 27.5 mm 胸苷的股票解决方案 (溶解在培养基中) 进入培养基与细胞最终浓度2.5 毫米胸苷。孵育的细胞, 在步骤3.1.1 为另 15 h. 冲洗和孵育的细胞在胸苷自由培养基4-6 小时, 并诱导 DNA 损伤 (4 节)。</li>
		<li>通过使用细胞周期标记 (分别为 S/G2 和 G2)、S/G2 相特异的 DNA 损伤修复因子 (例如、Rad51 或内聚力) 或 IdU/教育合并 (S 相细胞)51来确认同步效率。</li>
	</ol>
</li>
	<li>
G1 相位同步
	<ol>
<li>种子 6-8 x 104 HeLa 细胞在35毫米培养皿中与格片 (步骤 2.1)。</li>
		<li>2天后, 在倒置显微镜下使用10X 物镜和10X 眼透镜, 确定中期细胞, 这是轻微提升和圆形的。获取图像以记录中期细胞在格片上的位置。</li>
		<li>3-4 小时后, 确认细胞分裂成两个子细胞 (在 G1 阶段), 并诱导 DNA 损伤 (第4节)。</li>
	</ol>
</li>
</ol>4. 激光输入功率的滴定
注: 在本协议中, 我们使用 780 nm 脉冲 fs 近红外激光系统的共聚焦显微镜, 或 800 nm 脉冲 fs 近红外激光耦合到倒置的 epi 荧光显微镜诱导 DNA 损伤。虽然在这些系统中, 输入功率 (在试样的激光焦点的实际辐照度) 需要诱导可比较的 DDR 不同, 但输入功率滴定的基本原理适用于两个或任何一个近红外系统32,57. 在两个激光系统的焦点上, 每激光脉冲和峰值辐照度的原位能量分别在 5.33 x 10-2-4 x 10-1 新泽西和 7 x 10 10-5.24 x 10 11 W/cm2, 分别为32。
<ol><li>打开近红外激光器并允许激光系统在使用前预热1小时。</li>
	<li>在加热的 (37 ° c) 的腔室中放置一盘细胞, 允许 CO2 (5%) 和湿度 (100%) 控制。 注意: 如果 co2室不可用, 请使用 co2独立的区域性媒体, 最多5-6 小时。如果没有加热阶段, 保持显微镜下的细胞 < 20 分钟, 并立即更换到组织培养孵化器与适当的温度, CO2, 和湿度控制。这些培养条件可能因所使用的特定细胞类型以及派生 (例如、哺乳动物与两栖动物) 的有机体的不同而有所不同。</li>
	<li>对于荧光标记蛋白质的活细胞分析, 选择一个 gfp 过滤器 (450-490 nm 激发, 515-586 nm 发射) 或 Cy3 过滤器 (540-552 nm 激发, > 590 nm 发射) 的 gfp 或 mCherry 融合蛋白, 分别使用100X 或63X 油浸泡目的.</li>
	<li>搜索具有可比较荧光信号的细胞, 反映蛋白质表达的可比水平。避免细胞有太弱或太强的表达, 以减少细胞对细胞的变异荧光测量。</li>
	<li>
滴定激光输入功率。
	<ol>
<li>780纳米近红外激光在共焦显微镜上的附着<ol>
<li>使用软件漂白功能的目标是在细胞核内的线性轨道曝光到单一的激光扫描 (分辨率 512 x 512 像素, 变焦 X1, 漂白区域 50 x 4 像素, 12.8 µs 像素停留时间, 迭代 x1) 通过石油目标 (100 x/1.3 NA)。</li>
			<li>调整激光传输百分比 (使用由制造商在激光显微镜系统中内置的软件/硬件) 到 5%, 而不更改任何其他设置。</li>
			<li>将单元格置于该字段的中心。单击 "感兴趣区域 (ROI)" 工具, 然后使用鼠标在细胞核中绘制一条线或框 (大约 50 x 4 像素), 然后单击 "漂白" 按钮以诱发 DNA 损伤。 注: 在随后的辐照步骤中, "漂白" 应增加5% 增量, 以40% 或高达 100%, 如果需要。</li>
			<li>800 nm 近红外激光与 epi 荧光显微镜的连接</li>
			<li>控制输入功率通过一个 (63 x/1.4 NA) 相衬油浸泡目标由在光束路径引入的极化滤波器的机动旋转, 并安装在计算机控制的机动旋转阶段32,39,58。</li>
			<li>调整偏光角 (°), 并测量输入功率 (兆瓦) 进入显微镜, 使用激光功率计。确定提供输入功率范围从 20 mW-155 兆瓦在5-10 兆瓦增量的偏光器角度。</li>
			<li>设置与20兆瓦输入功率对应的偏光角。 注: 对于我们的激光系统, 如果偏光角为40°, 输入功率为65兆瓦。增加5-10 兆瓦增量的输入功率。</li>
		</ol>
</li>
	</ol>
</li>
	<li>对于活细胞分析, 请转到步骤6.1 和6.3。为免疫荧光检测内源性蛋白或修改进行5节。分析后, 转到步骤4.7 进一步滴定激光功率。要分析上游因素的要求, 请转到第7节。 注: BER 和 NHEJ 因素的招聘迅速 (在几分钟内) 和瞬态 (< 〜 30 min-1 h 取决于因素和 < 〜 4 h 后损伤诱导, 分别)。相比之下, HR 因素 Rad51 和内聚力的招募是可检测的在〜 30 min-1 h 和他们倾向于坚持超过8小时在未 DNA 损伤44,50,51。因此, 有必要检查不同的时间点后照射不同的修复因素。</li>
	<li>按预期的增量增加输入功率 (即, 5% 用于780近红外激光器和5-10 兆瓦的800的近红外线), 并在步骤4.4 中重复步骤 4.5-4.7, 在新的单元格上确定阈值 (50% 的受损细胞显示招募) 和峰值 (100% 的细胞显示招聘) 为每个蛋白质。</li>
</ol>5. 免疫荧光染色
注: 用于检测共价蛋白质修饰, 如 PAR (复合损伤标记) 和 H2AX 磷酸化 (γH2AX) (争端处理标记), 以及丁烷嘧啶二聚体 (6-4) 化 (6-4PP) 和 8-嘌呤 (8-oxoG) (交联和基底损伤标记分别), 细胞必须固定和染色的特定抗体。此外, 最好通过抗体检测内源性蛋白来确认荧光标记重组蛋白的结果, 以区分重组融合蛋白过度表达引起的伪影。
<ol><li>在损伤诱导后在不同的时间点固定细胞 (步骤 4.5) 取决于因素通过替换文化媒介 (参见步骤 1.1) 与1.5 毫升 4% paraformaldehyde/1X PBS 为 10 min 在 RT。 注意: 甲醛烟雾是有毒的, 所有的工作都应该在通风的油烟罩中进行。</li>
	<li>删除4% 甲醛/pbs 和1毫升0.5% 洗涤剂/pbs 在 RT 10 分钟。</li>
	<li>去除0.5% 洗涤剂/pbs 和冲洗细胞2毫升 pbs 5 分钟, 并孵育细胞在2毫升的阻断溶液 (10% 小牛血清, 1% BSA/PBS) 为1小时 RT。</li>
	<li>去除阻断液, 并将 pbs 的2毫升添加到细胞中, 用250µL 抗体溶液在 RT 的4° c 或1小时内将 pbs 替换为 3% BSA/pbs. 使用 1:200-1: 500 稀释 (典型的集中大约1µg/毫升, 经验主义地确定) 为抗体特定于不同的 DNA 损伤标记 (例如, γH2AX/53BP1 (争端分析);Ku (争端 NHEJ);Rad51/cohesin (争端管理, 人力资源);CPD/6-4PP (交联损伤);8-oxoG/DNA glycosylases (例如, NTH1) (基底损伤);PAR (高剂量复合损伤))。</li>
	<li>去除抗体溶液, 用2毫升 pbs 冲洗5分钟, 去除 pbs, 并在室温下的3% 小时内用250µL 0.1% 二次抗体孵育至 1 h。</li>
	<li>去除二次抗体溶液, 并增加2毫升 pbs 为5分钟两次, 并保持细胞在 1.5-2 毫升 pbs 在4° c 的成像步骤6.1 和6.2。</li>
</ol>6. Immunostained 或活细胞的定量荧光分析
<ol><li>使用用于照射的显微镜捕捉染色或活细胞的图像 (n > 10)。对于 epi 荧光显微镜系统, 使用分辨率为 1344 x 1024 像素, 并将图像保存为 TIFF 16 位文件。对于共焦显微镜, 使用1X 放大倍率;用于 GFP/FITC 的氩激光, 氖594激光用于 Cy3/mCherry;用于图像分辨率的 512 x 512 或 1024 x 1024 像素;扫描速度 5, 数字: 1 用于快速扫描或 2 +, 平均超过多个扫描, 以提高信噪比。</li>
	<li>
使用用于测量像素强度的图像分析程序。
	<ol>
<li>使用 "体积矩形" 工具在单个单元格图像中的损坏站点周围绘制 ROI。使用相同大小的 ROI 来测量核附近的背景荧光信号, 但不能与损伤部位重叠。这对于与非共焦显微镜系统相关的漂白的正常化非常重要。</li>
		<li>要使用图像分析程序测量荧光信号, 请单击工具箱中的 "音量分析报告"。将出现一个新窗口。在 "数据显示" 部分, 只检查 "名称" 和 "密度" 框。然后单击 "完成" 以可视化测量。</li>
		<li>通过单击在 "卷报告" 窗口底部找到的表格图标, 将这些值导出到电子表格程序中, 然后为 "导出目标" 字段分隔符和 "文件" 选择 "制表符 (excel 格式)"。</li>
		<li>为了获得相对的信号, 使用电子表格程序将损伤部位 (1 栏) 的荧光强度除以核的控制背景区域 (2 列), 然后减去1以进行正常化。</li>
	</ol>
</li>
	<li>
用共焦显微镜实时定量荧光时间-过程分析
	<ol>
<li>在步骤4.3 中使用显微镜识别荧光标记的蛋白质阳性细胞。</li>
		<li>在4节损伤诱导后, 在损伤前和不同时间点进行延时荧光成像。首先画出漂白剂的 ROI (对损伤感应), 然后选择 "获取 > 时间序列" 子菜单, 设置 "数字" 为 20, 和 "周期延迟" 为三十年代, 选择 "漂白一次后第一次扫描", 然后单击 "开始 B"。在这种情况下, 第一个图像是在损伤诱导前立即获得, 然后在三十年代的时间间隔内进行19次图像获取。时间间隔 ("周期延迟") 可以根据研究的目的而改变。</li>
		<li>一旦图像采集完成, 点击 "平均值", 并在损害区域绘制 ROI。单击 "显示表" 以获得一个表, 列出所选 roi 中的强度。通过将损伤部位的荧光强度除以同一区域在损伤前的强度, 使数据正常化, 并减去1。</li>
	</ol>
</li>
</ol>7. 小干扰 RNA (siRNA) 转染
注意: 目标蛋白的耗竭是描述被观察到的蛋白质招募到损伤部位的上游因素要求的有效方法 (4 节)。此外, 该战略是解决免疫荧光检测抗体特异性的最佳方法 (见5节)。
<ol><li>1天: 在24孔板上播种 6-8 x 104 hela 细胞, 在每口井的 0.5 mL 培养基中制备2口的 hela 细胞, 一个用于控制 siRNA 转染, 一个用于 PARP1 siRNA (5 CCG) TTA。</li>
	<li>2天: 确认细胞约40-50% 汇合。执行第一轮 siRNA 转染: 稀释 5 pmol siRNA 成25µL 无血清培养基, 加入3µL 脂质基转染试剂到试管中, 轻轻混合, 孵育5-10 分钟。</li>
	<li>用0.5 毫升新鲜培养基冲洗细胞一次, 在0.5 毫升新鲜培养基中保持细胞 (见步骤 1.1)。</li>
	<li>将 RNA 脂复合物添加到细胞中, 然后通过来回倾斜来轻轻混合。将单元格放回孵化器, 如步骤2.1 所示。</li>
	<li>吸入转染混合, 并添加0.5 毫升新鲜培养基 (步骤 1.1) 后6小时。</li>
	<li>3天: 执行第二轮 siRNA 染 (参见步骤 7.2)。然后, 种子60-100% 的细胞成35毫米格片盘, 如步骤2.3 所述。</li>
	<li>5天: 使用最佳激光功率条件进行激光 microirradiation, 以最大程度地招募感兴趣的因素, 如4节所确定的那样。通常, 在大约60-90% 的 HeLa 细胞中可以观察到有效的损耗。 注: 作为一种替代和互补的方法, 抑制剂, 如果可用, 可以用来抑制功能的因素的利益32,44。为分析抑制 DDR 信令的效果, 添加20µM PARP 抑制剂, 1 µM PAR glycohydrolase (PARG) 抑制剂, 或10µM dna 依赖性蛋白激酶与催化亚基 (dna-PKcs) 抑制剂和10µM ATM 抑制剂在二甲基亚砜 (砜) 的在损伤诱导前1小时的盘子。只将亚砜添加到控制单元中。</li>
</ol>

作者没有什么可透露的。

使用激光 microirradiation 进行 DDR 研究的优点是:
1. 从简单的链断裂到复杂的 dna 损伤, 可以诱导不同类型和数量的 dna 损伤, 并通过调整激光辐照参数来检测 dna 损伤部位的不同修复因子。也可以在相同的细胞核中多次造成损伤, 以评估反式效果 (如图 3所示)。
2. 重要的是, 在受损地点发生的事件和发生在原子核其他地方的次要事?...

在体内DNA 损伤信号不太清楚 在体内, DNA 与组蛋白和其他因素复合形成染色质纤维。染色质结构的调节对 DNA 代谢至关重要。例如, 组蛋白变体 H2AX 是由共济失调-扩张突变 (ATM) 和其他激酶后的双链断裂 (争端) 诱导磷酸化, 是重要的争端处理的损害信号放大, 以及提供一个对接地点的其他因素.损伤信号的传播和修复途径的选择似乎受到局部染色质结构的严重影响1。一些染色质重塑因子, 组蛋白的陪护, 和组蛋白修饰酶确实被招募到破坏地点, 是重要的有效的 DNA 修复, 突出了染色质调节的意义在 DDR 和修复2,3,4. 此外, 在酵母和果蝇5,6,7,8中观察到的损伤站点聚类或重新定位, 让人联想到孵育的基因座与基因调控相关的层次室9,10。最近在老鼠和人类细胞中的研究也显示了对争端修复点的动员, 这影响了维修保真度和路径选择11,12。这就增加了 DDR/修复也可能与核结构、高阶染色质组织和细胞核内的染色体动力学紧密相连的可能性。因此, 必须发展高分辨率的方法, 以研究在活细胞内核环境的背景下的 DDR 和修复过程, 以便了解 DNA 损伤的短期和长期后果。
PAR 聚合酶 (PARP) 在测量损伤部位的损伤程度和类型以及调节蛋白质组装方面的关键作用 PARP1 是 dna 尼克传感器迅速激活 dna 损伤, 在 dna 修复13中扮演关键角色。PARP1 最初被认为与 X 射线修复交叉配合 1 (XRCC1), 以促进基础切除修复 (BER), 但最近的研究显示它的作用在其他 DNA 修复途径, 包括争端修复14。活化 PARP1 使用烟酰胺腺嘌呤核苷酸 (和+) 作为基板, 以 ADP-ribosylate 多靶蛋白, 包括本身。近年来, 这种酶和其他家族成员引起了人们的广泛关注, 因为 PARP 抑制剂已经成为一种有前途的癌症治疗药物。虽然 PARP 抑制剂最初被发现是有效的乳腺癌基因 (BRCA)-突变乳腺癌细胞, 现在有大量的证据, 他们的影响, 在单一和联合治疗与 DNA 损伤的代理人/辐照广泛的癌症的突变不限于 BRCA15,16,17,1819。
在分子水平, PARP 活化被证明在组织地方染色质结构在损伤站点扮演重要角色。依赖于标准的染色质修饰酶的招募促进了争端修复, 并决定了修复路径的选择, 这表明了在损伤部位进行 par 修饰的重要脚手架作用。 13 ,21,22,,24,25,26,2728,2930,31我们最近演示了将 p53-binding 蛋白 1 (53BP1) 从损坏点排除在 PAR 32之外, 为非同源 endjoining 的53BP1 依赖 hyperactivation 提供了另一种解释 (NHEJ) 通过 PARP 抑制剂和突出 PARP 的意义在争端修复路径选择33,34。PARP1 也直接 PARylates 并影响多种 DNA 修复因子的活性14。
使用激光 Microirradiation 作为一种工具来研究 DDR/修复在体内 在 1969年35中首次描述了激光 microirradiation 在单个染色体上产生亚微米的变化, 并在1981年的36中进行了详细的回顾。几十年后, 激光 microirradiation 被证明是诱导 dna 损伤在一个明确的微米区域的细胞核, 并被证明是一个有价值的技术, 研究招募或修改各种因素的 dna 损害在体内13,37,38,39,40,41. 此方法允许检测未形成明显辐射诱发病灶 (IRIF) 在损伤部位的那些因素39,42。也可以在损伤部位和细胞核的其余部分研究染色质结构变化的时空动力学。我们仔细比较了不同激光系统诱导的 ddr 和输入功率, 以评估 DNA 损伤类型与 microirradiation 条件之间的关系32,43,44, 45。在先前的激光损伤研究中观察到了53BP1 和端重复约束因子 2 (TRF2) 的异常招聘模式, 这为激光损伤的 "非生理性" 特性的反复关注提供了依据46 ,47,48,49。我们发现, 这些明显的差异现在可以解释的差异 PARP 信号, 测量的数量和复杂的诱导损害32。我们证实: 1) 激光 microirradiated 细胞 (即使在高输入功率辐照后) 在界面中以损伤检查点控制的方式被逮捕, 并且保持可行 (至少 48 h)32,50;和 2) 修复因子的招募/修改忠实地重述那些观察与常规 DNA 损伤药物和争端处理酶32,39,42, 44、50、51、52。这些结果强烈支持研究激光损伤诱导的细胞反应的生理相关性。

<table><tbody><tr><td>Ti:Sapphire NIR pulsed femtosecond laser Mira-900</td><td>Coherent Inc.</td><td>Mira 900</td><td></td></tr><tr><td>Inverted microscope&amp;nbsp;</td><td>Carl Zeiss</td><td>Axiovert 200M</td><td></td></tr><tr><td>63X/1.4 NA objective</td><td>Zeiss</td><td>APOCHROMAT Ph3&amp;nbsp;</td><td></td></tr><tr><td>Compact Rotation Stage&amp;nbsp;</td><td>Newport Corp</td><td>PR50PP</td><td></td></tr><tr><td>Temperature Controller</td><td>Warner</td><td>TC-344B</td><td></td></tr><tr><td>Heating System</td><td>Ibidi&amp;nbsp;</td><td>10918</td><td></td></tr><tr><td>Gas Incubation System</td><td>Ibidi&amp;nbsp;</td><td>11920</td><td></td></tr><tr><td>Laser Power and Energy Meter (RoHS)</td><td>Coherent</td><td>FieldMaxII-TOP&amp;nbsp;</td><td></td></tr><tr><td>ORCA-R2 Digital CCD Camera</td><td>Hamamatsu</td><td>C10600</td><td></td></tr><tr><td>LSM 510 META Laser Scanning Microscopes</td><td>Carl Zeiss</td><td></td><td></td></tr><tr><td>100X/1.3 NA Zeiss Plan APO</td><td>Carl Zeiss</td><td></td><td></td></tr><tr><td>35mm Gridded Dishes&amp;nbsp;</td><td>MatTek</td><td>P35G-1.5-14-CGRD-D</td><td></td></tr><tr><td>PtK2 kidney epithelial cells</td><td>ATCC</td><td>CCL 56&amp;nbsp;</td><td></td></tr><tr><td>HeLa cells</td><td>ATCC</td><td>CCL-2</td><td></td></tr><tr><td>DMEM</td><td>Life Technologies</td><td>11885-092</td><td></td></tr><tr><td>CO2-Independent Medium&amp;nbsp;</td><td>Life Technologies</td><td>18045-088</td><td></td></tr><tr><td>Advanced MEM&amp;nbsp;</td><td>Life Technologies</td><td>12492-013</td><td>supplemented with L-Glutamine, 4% FBS</td></tr><tr><td>penicillin/streptomycin</td><td>Fisher Scientific</td><td>15140122</td><td></td></tr><tr><td>L-Glutamine</td><td>Fisher Scientific</td><td>25030081</td><td></td></tr><tr><td>FBS</td><td>Omega Scientific</td><td>FB-02</td><td></td></tr><tr><td>Thymidine&amp;nbsp;</td><td>&amp;nbsp;SIGMA</td><td>T9250</td><td></td></tr><tr><td>serum free media&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; (Opti-MEM I Reduced Serum Media)</td><td>Fisher Scientific</td><td>11058021</td><td></td></tr><tr><td>Anti-Cycolbutance pyrimidine dimer (CPD) (mouse)</td><td>Kamiya Biomedical Company</td><td>MC-062</td><td></td></tr><tr><td>Anti-XRCC1 (mouse )</td><td>Gene Tex Inc</td><td>GTX72311</td><td></td></tr><tr><td>Anti-53BP1 (rabbit)</td><td>Santa Cruz Biotech</td><td>sc-22760</td><td></td></tr><tr><td>Anti-CtIP (rabbit)</td><td>Abcam</td><td>ab70163</td><td></td></tr><tr><td>Anti-PAR polymers (mouse)</td><td>Enzo Life Sciences</td><td>BML-SA216-0100</td><td></td></tr><tr><td>Anti-PAR (rabbit)</td><td>Trevigen</td><td>4336-BPC-100</td><td></td></tr><tr><td>Anti-TRF2 (mouse)</td><td>Novus Biological&amp;nbsp;</td><td>NB100-56506</td><td></td></tr><tr><td>Anti 6-4PP (mouse)</td><td>Kamiya Biomedical</td><td></td><td></td></tr><tr><td>Anti-Rad21 (Rabbit)&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; (for cohesin detection)</td><td>generated in Yokomori (KY) lab</td><td></td><td></td></tr><tr><td>Anti-Rad51 (Rabbit)</td><td>Santa Cruz Biotechnology</td><td>SC-8349</td><td></td></tr><tr><td>Anti-Ku70 (mouse)</td><td>&amp;nbsp;Novus Biologicals</td><td>NB100-102</td><td></td></tr><tr><td>8-oxiguanine&amp;nbsp;</td><td>Trevigen, Inc.</td><td></td><td></td></tr><tr><td>Anti-PARP1&amp;nbsp; (Rabbit)&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>generated in KY lab</td><td></td><td>ref 43</td></tr><tr><td>&amp;nbsp;Anti-hCAPG (Rabbit)&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; (for condensin detection)</td><td>generated in KY lab</td><td></td><td>ref 42</td></tr><tr><td>Cy3 AffiniPure Donkey Anti-Rabbit IgG</td><td>&amp;nbsp;Jackson ImmunoResearch Inc</td><td>711-165-152</td><td></td></tr><tr><td>Donkey Anti-Mouse Alexa Fluor 488 IgG</td><td>Thermo Fisher Scientific</td><td>A-21202</td><td></td></tr><tr><td>HiPerFect siRNA Transfection Reagent</td><td>Qiagen</td><td>301705</td><td></td></tr><tr><td>Lipofectamine 2000 Transfection Reagent</td><td>Thermo Fisher Scientific</td><td>11668019</td><td></td></tr><tr><td>GFP-SMC1 stable cell line&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>generated in KY lab</td><td></td><td>ref 49&amp;nbsp;</td></tr><tr><td>GFP-NEIL2 stable cell line</td><td>generated in KY lab</td><td></td><td>ref 54</td></tr><tr><td>GFP-NTH1 stable cell line</td><td>generated in KY lab</td><td></td><td>ref 32</td></tr><tr><td>GFP-SMC1 plasmid</td><td>generated in KY lab</td><td></td><td></td></tr><tr><td>GFP-Ku plasmid</td><td>generated in KY lab</td><td></td><td></td></tr><tr><td>Anti-MDC1 (rabbit)</td><td>&amp;nbsp;Novus Biologicals</td><td>NB100&amp;ndash;395</td><td></td></tr><tr><td>Anti-gH2AX (rabbit)</td><td>&amp;nbsp;Millipore</td><td>07&amp;ndash;164</td><td></td></tr><tr><td>EGFP-53BP1(1220-1711) PtK2 stable cell line</td><td>generated in Berns lab</td><td></td><td>ref 32</td></tr><tr><td>TRF2-YFP PtK2 stable cell line</td><td>generated in Berns lab</td><td></td><td>ref 32</td></tr><tr><td>DMSO</td><td>Sigma</td><td>D2650-100ML</td><td></td></tr><tr><td>PARG inhibitor</td><td>Trevigen</td><td>4680-096-03</td><td></td></tr><tr><td>DNA&amp;ndash;PKcs inhibitor&amp;nbsp; NU7026&amp;nbsp;</td><td>Sigma</td><td>N1537</td><td></td></tr><tr><td>ATM inhibitor KU55933</td><td>&amp;nbsp;Calbiochem</td><td>118500</td><td></td></tr><tr><td>Olaparib&amp;nbsp;</td><td>Apexbio Technology</td><td>A4154</td><td></td></tr><tr><td>Paraformaldehyde&amp;nbsp;</td><td>ELECTRON MICROSCOPY SCIENC MS</td><td>100503-916 (EA)</td><td></td></tr><tr><td>Quantity One 1-D Analysis Software Version 4.6.9</td><td>Bio-Rad</td><td>SOFT-LIT-70-9600-Q1-469PC&amp;nbsp;</td><td>image analysis program</td></tr><tr><td>Excel</td><td>microsoft</td><td></td><td>spreadsheet program</td></tr><tr><td>Triton X100</td><td>Fisher Scientific</td><td>BP151-500</td><td>detergent</td></tr><tr><td>Dulbecco&#039;s phosphate-buffered saline (DPBS) 10X without calcium and magnesium</td><td>Fisher Scientific</td><td>14200166</td><td>dilute to 1X&amp;nbsp;</td></tr></tbody></table>

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

DNA 损伤诱导细胞的特定信号和修复反应, 这对保护基因组完整性至关重要。激光 microirradiation 成了研究 DNA 损伤反应 (DDR)体内的有价值的实验工具。它允许实时高分辨率的单细胞分析大分子动力学, 以响应激光诱导的损伤局限于微米区域的细胞核。然而, 不同的激光条件已被使用, 不了解不同类型的损害引起的差异。因此, 损害的性质往往没有很好的特征或受到控制, 造成征聘或修改资料中明显的不一致。我们证明, 不同的辐照条件 (即, 不同的波长, 以及不同的输入功率 (度) 的飞秒 (fs) 近红外激光) 诱导不同的 DDR 和修复蛋白组件。这反映出 DNA 损伤的类型。该协议描述了如何滴定激光输入功率, 使不同数量和复杂的 DNA 损伤, 可以很容易地监测通过检测基地和交联损害, 差异聚 (ADP-核糖) (PAR) 信号,损伤部位的通路特定修复因子组件。一旦确定了损伤条件, 就有可能研究不同损伤复杂度和差异损伤信号的影响以及上游因素对任何感兴趣因素的损耗。

使用 PtK2 细胞稳定表达 EGFP-53BP11220-1711或 TRF2-YFP, 进行激光输入功率滴定, 以确定他们的最佳招聘条件 (图 1)32。
在高输入功率激光损伤现场, 明显的 GFP-NTH1 (紫外线) 损伤引起的交联损伤, 以及专门识别基底损伤的 DNA glycosylase 的显著聚类。此外, 观察到较高的 XRCC1 和 CtIP 信号, 反映了链断裂数量的增加, 并证明在这种情况下引起复...

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

该协议的目的是描述如何使用激光 microirradiation 诱导不同类型的 dna 损伤, 包括相对简单的断链和复杂的损害, 研究 dna 损伤信号和修复因子在损伤部位的组装在体内.

激光 Microirradiation 研究体内细胞对简单复杂 DNA 损伤的反应

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

Research

JoVE Journal

Genetics

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

10.2K 次观看.  University of California, Irvine. 该协议的目的是描述如何使用激光 microirradiation 诱导不同类型的 dna 损伤, 包括相对简单的断链和复杂的损害, 研究 dna 损伤信号和修复因子在损伤部位的组装在体内.

视频: 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.

植物病原体的遗传操作<em&gt;玉米黑粉菌</em&gt;研究真菌生物学和植物微生物相互作用

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 ...

科研

遗传学

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.

工具来研究蛋白质建筑HMGB1的作用，螺旋窜改，站点特定的DNA间交联处理

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.

肽衍生的方法在运输跨越植物细胞的基因和蛋白质和细胞器障碍

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 is by far the most common pathway to initiate protein synthesis in eukaryotic cells, but stress-specific variations of this complex are now emerging. Purifying cap-binding proteins with an affinity resin composed of Agarose-linked m7GTP (a 5' mRNA cap analog) is a useful tool to identify factors involved in the regulation of translation initiation. Hypoxia (low oxygen) is a cellular stress encountered during fetal development and tumor progression, and is highly dependent on translation regulation. Furthermore, it was recently reported that human adult organs have a lower oxygen content (physioxia 1-9% oxygen) that is closer to hypoxia than the ambient air where cells are routinely cultured. With the ongoing characterization of a hypoxic eIF4F complex (eIF4FH), there is increasing interest in understanding oxygen-dependent translation initiation through the 5' mRNA cap. We have recently developed a human cell culture method to analyze cap-binding proteins that are regulated by oxygen availability. This protocol emphasizes that cell culture and lysis be performed in a hypoxia workstation to eliminate exposure to oxygen. Cells must be incubated for at least 24 hr for the liquid media to equilibrate with the atmosphere within the workstation. To avoid this limitation, pre-conditioned media (de-oxygenated) can be added to cells if shorter time points are required. Certain cap-binding proteins require interactions with a second base or can hydrolyze the m7GTP, therefore some cap interactors may be missed in the purification process. Agarose-linked to enzymatically resistant cap analogs may be substituted in this protocol. This method allows the user to identify novel oxygen-regulated translation factors involved in cap-dependent translation.

Here, we present human cell culture protocols to analyze translation initiation factors that bind the 5' cap of mRNA during physiological oxygen conditions. This method utilizes an Agarose-linked m7GTP cap analog and is suitable to investigate cap-binding factors and their interacting partners.

暴露在生理条件下的氧气在人体细胞中帽结合蛋白质分析

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

Single Molecule Analysis of Laser Localized Psoralen Adducts

The DNA Damage Response (DDR) has been extensively characterized in studies of double strand breaks (DSBs) induced by laser micro beam irradiation in live cells. The DDR to helix distorting covalent DNA modifications, including interstrand DNA crosslinks (ICLs), is not as well defined. We have studied the DDR stimulated by ICLs, localized by laser photoactivation of immunotagged psoralens, in the nuclei of live cells. In order to address fundamental questions about adduct distribution and replication fork encounters, we combined laser localization with two other technologies. DNA fibers are often used to display the progress of replication forks by immunofluorescence of nucleoside analogues incorporated during short pulses. Immunoquantum dots have been widely employed for single molecule imaging. In the new approach, DNA fibers from cells carrying laser localized ICLs are spread onto microscope slides. The tagged ICLs are displayed with immunoquantum dots and the inter-lesion distances determined. Replication fork collisions with ICLs can be visualized and different encounter patterns identified and quantitated.

Lasers are frequently used in studies of the cellular response to DNA damage. However, they generate lesions whose spacing, frequency, and collisions with replication forks are rarely characterized. Here, we describe an approach that enables the determination of these parameters with laser localized interstrand crosslinks.

激光局部补骨脂素加合物的单分子分析

The DNA Damage Response (DDR) has been extensively characterized in studies of double strand breaks (DSBs) induced by laser micro beam irradiation in live ...

Application of Laser Micro-irradiation for Examination of Single and Double Strand Break Repair in Mammalian Cells

Highly coordinated DNA repair pathways exist to detect, excise and replace damaged DNA bases, and coordinate repair of DNA strand breaks. While molecular biology techniques have clarified structure, enzymatic functions, and kinetics of repair proteins, there is still a need to understand how repair is coordinated within the nucleus. Laser micro-irradiation offers a powerful tool for inducing DNA damage and monitoring the recruitment of repair proteins. Induction of DNA damage by laser micro-irradiation can occur with a range of wavelengths, and users can reliably induce single strand breaks, base lesions and double strand breaks with a range of doses. Here, laser micro-irradiation is used to examine repair of single and double strand breaks induced by two common confocal laser wavelengths, 355 nm and 405 nm. Further, proper characterization of the applied laser dose for inducing specific damage mixtures is described, so users can reproducibly perform laser micro-irradiation data acquisition and analysis.

Confocal fluorescence microscopy and laser micro-irradiation offer tools for inducing DNA damage and monitoring the response of DNA repair proteins in selected sub-nuclear areas. This technique has significantly advanced our knowledge of damage detection, signaling, and recruitment. This manuscript demonstrates these technologies to examine single and double strand break repair.

激光微辐照在哺乳动物细胞单、双链断裂修复中的应用

Highly coordinated DNA repair pathways exist to detect, excise and replace damaged DNA bases, and coordinate repair of DNA strand breaks. While molecular ...

癌症研究

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.

低丰度胚胎标本的染色质沉淀 (芯片) 协议

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

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.

405 Nm 激光微辐照对 DNA 损伤的蛋白质招募研究

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

Advanced Confocal Microscopy Techniques to Study Protein-protein Interactions and Kinetics at DNA Lesions

Local microirradiation with lasers represents a useful tool for studies of DNA-repair-related processes in live cells. Here, we describe a methodological approach to analyzing protein kinetics at DNA lesions over time or protein-protein interactions on locally microirradiated chromatin. We also show how to recognize individual phases of the cell cycle using the Fucci cellular system to study cell-cycle-dependent protein kinetics at DNA lesions. A methodological description of the use of two UV lasers (355 nm and 405 nm) to induce different types of DNA damage is also presented. Only the cells microirradiated by the 405-nm diode laser proceeded through mitosis normally and were devoid of cyclobutane pyrimidine dimers (CPDs). We also show how microirradiated cells can be fixed at a given time point to perform immunodetection of the endogenous proteins of interest. For the DNA repair studies, we additionally describe the use of biophysical methods including FRAP (Fluorescence Recovery After Photobleaching) and FLIM (Fluorescence Lifetime Imaging Microscopy) in cells with spontaneously occurring DNA damage foci. We also show an application of FLIM-FRET (Fluorescence Resonance Energy Transfer) in experimental studies of protein-protein interactions.

Laser microirradiation is a useful tool for studies of DNA repair in living cells. A methodological approach for the use of UVA lasers to induce various DNA lesions is shown. We have optimized a method for local microirradiation that maintains the normal cell cycle; thus, irradiated cells proceed through mitosis.

高级共焦显微镜技术研究 DNA 损伤的蛋白质-蛋白质相互作用和动力学

Local microirradiation with lasers represents a useful tool for studies of DNA-repair-related processes in live cells. Here, we describe a methodological ...

生物学

Laser Micro-Irradiation to Study DNA Recruitment During S Phase

DNA damage repair maintains the genetic integrity of cells in a highly reactive environment. Cells may accumulate various types of DNA damage due to both endogenous and exogenous sources such as metabolic activities or UV radiation. Without DNA repair, the cell's genetic code becomes compromised, undermining the structures and functions of proteins and potentially causing disease. 
Understanding the spatiotemporal dynamics of the different DNA repair pathways in various cell cycle phases is crucial in the field of DNA damage repair. Current fluorescent microscopy techniques provide great tools to measure the recruitment kinetics of different repair proteins after DNA damage induction. DNA synthesis during the S phase of the cell cycle is a peculiar point in cell fate regarding DNA repair. It provides a unique window to screen the entire genome for mistakes. At the same time, DNA synthesis errors also pose a threat to DNA integrity that is not encountered in non-dividing cells. Therefore, DNA repair processes differ significantly in S phase as compared to other phases of the cell cycle, and those differences are poorly understood. 
The following protocol describes the preparation of cell lines and the measurement of dynamics of DNA repair proteins in S phase at locally induced DNA damage sites, using a laser-scanning confocal microscope equipped with a 405 nm laser line. Tagged PCNA (with mPlum) is used as a cell cycle marker combined with an AcGFP-labeled repair protein of interest (i.e., EXO1b) to measure the DNA damage recruitment in S phase.

This protocol describes a non-invasive method to efficiently identify S-phase cells for downstream microscopy studies, such as measuring DNA repair protein recruitment by laser micro-irradiation.

激光微辐射研究S阶段DNA招募

DNA damage repair maintains the genetic integrity of cells in a highly reactive environment. Cells may accumulate various types of DNA damage due to both ...