这项工作得到了加拿大自然科学和工程研究委员会 (5026 至 a. m) 和由来自癌症研究协会的下一代科学家奖学金 (21531 至 a. m) 的支持。我们感谢吉恩-弗朗索瓦 Lucier 为斐济 Python 脚本提供了卓越的质量控制, 并对统计分析提出了建议。我们感谢斯蒂芬妮博士 a Yazinski 的固定溶液配方。我们感谢保罗-朱利 Karsenti 的帮助与激光功率测量。

1. 细胞的预敏化
<ol><li>24 h 在微辐照实验之前, 4万 U2OS 细胞每井在 1x DMEM 包含10% 个血清在8井养殖幻灯片与170µm 厚实盖玻片象玻璃或聚合物底部保证最大透射率405毫微米激光光和卓越的成像激光扫描倒置共焦显微镜。如果使用8井 microslide, 则可以使用500µL 的介质或解决方案, 为以后的所有处理和清洗该协议的步骤。 注意: 由于其扁平的形态, 良好的粘附性和较大的细胞核, U2OS 细胞有助于检测在微辐照部位的蛋白质的招募, 但其他黏附细胞类型可以根据需要使用。在微辐照时, 细胞最好是大约80% 融合, 以增加每个实验辐照细胞的数量。非常高的汇流可能导致细胞周期的改变, 可以扰乱特定的修复途径, 如同源重组, 发生在 S 和 G2 阶段的细胞周期9。</li>
	<li>用 5% CO2在37摄氏度的夜间孵化细胞, 然后用 (步骤 1.3) BrdU 或 (步骤 1.4) BBET (赫斯特 33342) 对细胞进行预敏。 注: 观察对 dna 病变、染或传感器质粒 dna 进行活体招聘, 并在细胞播种后携带荧光蛋白与24小时之间的融合。24小时后转染/转导, 预敏和微照射细胞, 以监测在 DNA 损伤网站的招聘, 实时。</li>
	<li>为了增加由博览会产生的 DSBs 405 nm 激光器的数量, 在微辐照前添加10µM 的 BrdU 到介质中24小时。在微辐照前, 使用微, 用培养基冲洗两次细胞而不含酚红, 以确保细胞最大程度地暴露于405纳米激光。</li>
	<li>或者, 在37摄氏度的细胞培养孵化器的适当培养基中, 用 BBET 在10µg/毫升上治疗细胞15分钟。治疗后, 在微辐照前用培养基冲洗两次, 无酚红。</li>
</ol>2. 细胞微照射
<ol><li>选择合适的激光扫描共聚焦显微镜。 注: 本文所做的实验是在显微镜系统上进行的, 配备50兆瓦405纳米激光线和 63X 1.4 的数字孔径油目标。为了生成 DSBs, 细胞在1X 扫描时被微辐照, 放大 1024 x 1024 像素/场 (0.21 µm/像素63X 放大), 在8µs/像素的单向模式下进行。所使用的激光功率分别为 BBET 和 BrdU 预敏细胞的25% 和80%。为帮助用户配置系统, 根据制造商的指示, 使用功率计测量了所提出系统上的激光电源后目标。测量结果分别对应于2.6 兆瓦和7.0 兆瓦的 BBET 和 BrdU 预敏细胞。</li>
	<li>
打开显微镜和环境室。
	<ol>
<li>将腔室设置为37°c, 5% CO2 , 然后将样品放入舞台孵化器中, 以避免对细胞造成不必要的压力, 并确保在实验过程中均匀的 DDR 动力学。</li>
	</ol>
</li>
	<li>
选择具有分布良好的单元格的字段, 调整焦点, 并注册它们的位置以便于在 IF 协议之后获取图像。获取至少一个图像, 作为前损伤参考点, 以监测微辐照后蛋白质招募的动力学。 注: 网格化培养容器也可用于在后续步骤中促进微辐照细胞的定位。<ol>
<li>如果使用 BBET 标记的细胞进行实验, 用 405 nm 激光在最低的激光功率下调整细胞的聚焦, 就足以使细胞形象化, 以避免 DNA 的不合理损伤。<ol>
<li>避免使用 widefield 荧光调节 BBET 染色细胞的焦点, 因为灯发出的紫外线光会诱发光照部位的 DNA 损伤。</li>
		</ol>
</li>
		<li>如果使用 BrdU 标记的细胞, 用差分干涉对比 (DIC) 或相位对比成像来调整焦点, 方法是观察核仁等亚核特征。</li>
		<li>如果使用表达荧光标记 POIs 的细胞, 请选择具有代表性荧光强度的焦平面。 注意: 避免细胞表现出很高的浓度, 因为强烈的过度表达可能导致 artifactual 的定位。另外, 在可比较的水平上选择表达兴趣的单元格 (强烈建议选择稳定的克隆, 以尽量减少兴趣表达式和招聘级别的变化)。</li>
	</ol>
</li>
	<li>利用系统漂白 (酶) _module 后的荧光恢复, 为微辐照步骤配置显微镜。 注: 在建立微辐照实验显微镜系统时要考虑的最重要参数是: 405 nm 激光器的输出功率, 迭代次数 (即,激光将在同一坐标上的次数),像素/字段数 (在63X 放大倍数和 1024 x 1024 分辨率, 1 像素 = 0.21 µm) 和每个像素的停留时间。所有这些因素都会影响细胞所经历的能量量, 从而产生 DNA 损伤的数量和类型 (请参见讨论以获得进一步的剂量优化信息)。</li>
	<li>利用显微镜系统的酶模块, 对细胞进行微照射。 注意: 在大多数系统中, 多单元格的微辐照可以在单个字段中进行, 如斑点或线/矩形 (通常为1-2 µm 宽)。当对荧光标记的蛋白质进行实时成像后立即损伤后, 将微辐射限制在每个领域的少量细胞中, 因为每细胞微照射的时间将导致复员方案的延迟诱导。这在监测对 DNA 损伤非常迅速地被吸收的蛋白质时尤为重要。</li>
	<li>
微辐照后, 在处理样本 (5 节) 或立即进行实时成像 (3 节) 之前, 在37°c 的孵化器中, 将多井幻灯片或板块放回 5% CO2 , 以达到所需的持续时间。
	<ol>
<li>作为第一步, 在几分钟内修复单元格, 并在照射后几个小时后进行 IF 协议。根据问题的不同, 招聘动力学可能会有所不同。通常, 在 DSBs 和办学团体的染色质相关的蛋白质会很快地定位到病灶, 有时在5分钟内被移除。一些因素, 如组装在 ssDNA 将被招募在以后的时间点, 一旦切除机械已经从事和保持在那里几个小时 (请参见图 1和参考4)。</li>
	</ol>
</li>
</ol>3. 荧光标记蛋白的活体成像
<ol><li>对微辐照细胞进行时间推移成像。对于非常迅速地被招募到病变的蛋白质, 每帧几秒钟是理想的, 但对于局部化到受损区域的蛋白质 (例如, 切除因子), 在达到高原之前就可以进行每分钟的采集。 注: 尽量减少图像采集的激光功率, 以避免尽可能漂白, 并确保在损伤部位的蛋白质招募不达到饱和, 这将妨碍信号量化和后续分析 (参见数据分析4节)。在所提出的系统上, 用 63 x/1.4 油目标进行成像, 使用8µs/像素停留时间, 1024 x 1024 分辨率, 1X 变焦, 平均2。请注意, 这些参数可能需要针对其他系统和/或不同的实验进行优化。</li>
	<li>微照射的细胞在额外的领域, 直到动力学测量获得15-30 细胞。在生物复制中重复实验至少3次, 以确保招聘动力学的准确性和重现性。 注意: 在将此协议修改为特定的共焦系统时, 从监视已知的荧光标记的基因组维护因子 (例如、53BP1 GFP、RPA32-GFP、等) 的招聘动力学开始可能很有用。使用实时成像优化微辐射条件, 用户可以快速测试多个参数。</li>
</ol>4. 招聘动力学分析
<ol><li>在斐济图像分析软件11中安装 Microirradiation 分析插件10 。</li>
	<li>
在斐济打开获取的图像。从 Microirradiation 分析套件的下拉插件菜单中选择 "损坏分析器"。按照提示定义感兴趣的背景区域 (ROI) (通常为 3 x 3 µm), 以及在每个微辐照单元格中未辐照和辐照的 ROIs。
	<ol>
<li>使用相同大小的 ROIs 来最小化平均信号强度中的潜在偏差。对于获得的图像, 也包括至少一个预辐照框架, 将作为基线, 以监测在微辐照网站的招聘。</li>
		<li>一旦所有 ROI 数据都已在全时移系列中收集, 请将包含结果的插件创建的文件保存为 csv (逗号分隔文件) 或 txt 文件 (制表符分隔文件)。该文件包含测量每个时间点的平均强度和每个 ROI (平均强度背景, 平均强度不照射, 平均强度照射)。 注意: 插件自动从辐照的 ROIs (is) 和未辐照的 ROIs (in) 的强度中减去背景强度 (Ib)。它还计算两个值之间的比率 (见下面的公式), 以纠正漂白 (辐照/未辐照)。 <img alt="Equation 1" src="/files/ftp_upload/57410/57410eq1v2.jpg"> 该插件还计算了第一个时间点 (值设置为 1) 和每个原子核的所有其他时间点之间的正常化 (相对于第一时间点的比率)。其他信息可以在插件网站10上找到。</li>
	</ol>
</li>
	<li>
使用插件对所有微辐照细胞 (至少15–30细胞) 进行分析, 并在一段时间内在微辐照区域内绘制平均相对富集。计算每个数据点的平均值的标准误差并绘制它们。
	<ol>
<li>通过对每个数据点执行学生的t测试, 确认两个实验条件之间的招聘动力学差异。</li>
	</ol>
</li>
</ol>5. 如果议定书
<ol><li>
准备解决方案。准备预/后萃取液 (含 0.25% X-100 的冰冷 1x pbs), 洗涤液 (1x pbs 含 0.05% Tween-20), 阻断溶液 (1x pbs 含 3% BSA 和 0.05% Tween-20), 固定溶液 (多聚甲醛 3% + 蔗糖2%在 PBS 1x) 在锥形聚丙烯管。 注: 对于单8井 microslide, 10 毫升的预/后萃取液, 50 毫升的洗涤液, 10 毫升的阻断溶液, 5 毫升的固定溶液是必需的。<ol>
<li>准备固定液。<ol>
<li>在一个化学罩下, 混合400毫升的双蒸馏水 (ddH2O) 与15克多聚甲醛和30µL 10 N 氢氧化钠在 1 L 锥形瓶。</li>
			<li>在搅拌溶解多聚甲醛的同时, 将容器和热在65°c 的热板上关闭。</li>
			<li>使用25毫升吸管, 添加50毫升 10X PBS, 搅拌和过滤器使用0.45 µm 过滤器。</li>
			<li>添加10克蔗糖和完整到500毫升与 ddH2O。</li>
			<li>整除15毫升圆锥管溶液;存储在-20 °c, 直到使用。</li>
		</ol>
</li>
		<li>在 1x PBS 中稀释 46-Diamidino 2-苯基吲哚 (DAPI) 1:1、000的1毫克/毫升的库存溶液, 制备核染色溶液。</li>
	</ol>
</li>
	<li>使用微, 小心地将介质从多井显微切片或板材的水井中取出, 并立即用500µL 冲洗溶液, 如果不等待就更换两次溶液。 注: 如果此处提供的数据是使用在所有解决方案的500µL 中冲洗和孵化的8井显微切片中生长的细胞获得的。卷应根据实验者使用的细胞培养容器进行调整。另外, 当细胞可以很容易地从玻璃/聚合物底部分离时, 执行 IF 协议以极端关心。使用微进行所有解决方案更改, 以最小化单元格丢失。</li>
	<li>在冰上孵育5分钟, 500 µL 的冰寒, 如果预/后萃取溶液和漩涡轻轻地用手 15–30 s 执行前提取步骤, 去除大多数细胞质和 nucleoplasmic 蛋白, 并使信号从染色质/与 DNA 相关的因素。</li>
	<li>洗两次, 500 µL, 如果洗涤解决方案。</li>
	<li>在室温下孵育15分钟, 在500µL, 如果固定溶液。或者, 为固定步骤使用预制作的多聚甲醛解决方案 (3–4%)。</li>
	<li>洗两次, 500 µL, 如果洗涤解决方案。</li>
	<li>在冰上孵育5分钟, 在500µL 的冰冷, 如果前/后萃取解决方案和漩涡的幻灯片轻轻地用手来执行通透步骤。</li>
	<li>洗两次, 500 µL, 如果洗涤解决方案。</li>
	<li>在室温下孵育至少30分钟, 如果阻塞溶液为500µL。</li>
	<li>使用微去除阻塞溶液, 在4摄氏度的湿润室中孵化过夜, 250 µL 主要抗体稀释在阻塞溶液中。 注意: 用主抗体顺序进行孵化是很好的做法, 以避免潜在的协同定位工件。一旦进行了适当的控制, 同时孵化出主要的抗体来加速这个过程。</li>
	<li>洗四次5分钟在500µL, 如果洗涤解决方案与温和搅拌 (60 rpms 在一个转子)。</li>
	<li>孵育1小时在37°c 与250µL 的二级抗体稀释在堵塞溶液中的湿化室。 注: 在添加荧光标记的二级抗体时, 保护样品不受光照。</li>
	<li>洗四次, 每5分钟在500µL, 如果洗涤溶液与温和搅拌。</li>
	<li>在室温下孵育5分钟, 500 µL 1x PBS, 含1µg/毫升 DAPI 以染色细胞核。</li>
	<li>用500µL 1x pbs 清洗两次, 并在500µL 1x pbs 溶液中留下细胞进行成像。</li>
	<li>使用网格化幻灯片或已注册的舞台位置, 使用具有良好特征的 DNA 损伤标记(例如、γ H2A X、RPA32 、等等) 找到微辐照的细胞。</li>
	<li>使用激光扫描共聚焦显微镜上的适当设置, 在所有相关的通道中成像多井文化幻灯片或板块。 注: 在所提出的系统, 成像是做了一个 63 x/1.4 油目标使用8µs/像素停留时间, 1024 x 1024 分辨率, 1X 变焦, 平均2。请注意, 这些参数可能需要针对其他系统和/或不同的实验进行优化。</li>
</ol>

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作者没有什么可透露的。

使用上面概述的方法, 405 纳米激光微辐照的细胞预敏与 BBET 或 BrdU 允许局部生成 DNA 病变, 包括 DSBs 在细胞核内黏附人细胞。DDR 在这些 DSBs 的动力学类似于在真核细胞中生成的其他方法9,18,19。在微辐照部位进行的肿瘤切除术可导致 ssDNA 的产生, 可以采用 RPA32-targeting 抗体或 RPA32-GFP 融合。与染色质相关的因素, 另一方面, 可以定位使用γ H2A. X 作为参考。一些染色质相关因素可能不会像γ H2A 一样扩散到最初的争端发生点. X 并且将出现作为有斑点的病灶, 可能有别于 ssDNA 结合蛋白病灶。
一旦 DSBs 被生成, 蛋白质的招募和清除从损伤地点可以监测, 如果在固定的细胞。执行这些实验只需要一个合适的, 如果抗体反对的景点。如果不相容的抗体是不可用的, 质粒可以设计携带的基因的框架与特定的表位 (如: 标志, 他的标记) 或荧光蛋白。将这些质粒的瞬态或稳定转染/转导到粘附细胞, 可以用来确定是否有一个特定的因素被积极地招募到受损的 DNA。过度表达蛋白质可能导致错误定位, 并可能人为地提高他们的招募到微辐照条纹。为了获得更具代表性的内源蛋白的结果, CRISPR-Cas9 技术现在可以经常使用适当的表位20,21来标记任何感兴趣的基因。
在微辐射分析中提供的 Python 脚本斐济插件促进了 DDR 蛋白动力学的可视化、呈现、发布和分析。该分析包是开放源码和功能, 无论用显微镜平台进行微辐照实验和图像采集。所有需要的是, 在显微镜平台上拍摄的图像被保存为生物格式兼容文件22。由于背景、漂移和漂白矫正被自动执行, 在 DNA 损伤站点的蛋白质水平分析与提供的脚本是直接和非常快速的与典型的手工信号量化执行与显微图像采集软件。
如《议定书》所述, 用户必须仔细配置各自的激光扫描共焦显微镜系统, 以达到适当的损伤程度, 并诱发 DSBs, 遵循正常的 DDR 动力学。事实上, 微辐照产生的 DNA 病变的数量和类型将取决于激光波长和剂量以及预敏方法。用户需要记住, 即使 DSBs 是由这里描述的条件产生的, 405 纳米激光微辐照光敏细胞也会产生其他 DNA 病变的混合物, 包括 cyclopyrimidine 脂肪酸, 办学团体和氧化基地, 将不同的显微镜系统和设置23,24,25。因此, 需要对微辐照部位的病灶进行仔细监测, 以全面了解所产生的损伤和引起的反应。重要的是, 为了研究对 DSBs 的反应, 每个显微镜系统都应该通过监测标准 DNA 损伤蛋白的动力学和微辐照部位的平移后修饰来优化。例如, γ H2A X 和 RPA 复杂亚基 (RPA70、RPA32 或 RPA14) 可以方便地使用, 因为它们很容易检测并在 DSBs 快速堆积。作为一个经验法则, γ H2A 的 X 信号应该在早期 (5 分钟) 和晚 (高达 3–4 h) 的时间点后被可视为条纹, 而爱国军应该开始积聚为点状病灶, 大约10-15 分钟后微照射。如果观察到泛核γ H2A 信号, 细胞所接收的能量是非生理的, 会导致细胞的快速死亡。
在使用 405 nm 激光进行特定 DNA 修复通路的研究时, 还需要考虑一些局限性。例如, 虽然 BBET-405 nm 组合容易产生办学团体和 DSBs 的组合, 但它不会产生 6–4 photoproducts (6–4 PPs), 因此, 核苷酸切除修复蛋白的招募动力学不同于那些观察使用紫外线-C 微辐照。在对 DSBs 的反应中所涉及的各种蛋白质的招募的动力学和程度也可能因使用23的 DNA 损伤方法而异。由于这些变化, 我们建议用户调整他们的系统, 以最大限度地扩大可能被他们的 POIs 识别的病变类型。虽然这种系统的范围不大, 但使用其他类型的激光器, 包括 UV A (337–355 nm) 或飞秒近红外线 (800 nm) 以及不同的敏化试剂 (例如: 三甲基补骨脂) 可以让用户生成更多特定的 dna 损伤取决于研究中的 dna 修复路径24,26。
在测试新的条件时, 使用荧光标记的成熟的 DNA 修复蛋白来测试剂量、波长和预敏剂组合是很方便的, 并确保有效地产生感兴趣的病变。例如, PARP1 和 XRCC1 可以作为单边带和基底切除修复的标志物, XPA 是核苷酸切除修复的一个很好的指标, 53BP1 将被招募到 DSBs 和在非同源端连接中的功能 (引用23,25 ,27,28)。识别与特定类型 dna 损伤相关的修饰的抗体也可以用来量化 dna 加合物或断裂的水平。因此, 有可能监测氧化碱, cyclopyrimidine 脂肪酸, 6–4 PPs, 办学团体 (使用聚 ADP 核糖抗体), DSBs (γ H2A. X 和 53BP1) 通过 IF 和确定的参数, 将达到最高水平的预期病变 23,27,29. 经过仔细的监控, 用户应该能够利用微辐射来研究其修复途径。
总之, 采用宽扩激光扫描共聚焦显微镜, 配备405纳米激光线, 产生局部 DSBs, 结合平台特定的 DNA 损伤标记, 对基因组维持因子进行分类, 并简化数据在微辐射分析的帮助下处理斐济插件, 应该使对 DDR 因素动力学的研究比以往任何时候都更容易接触到基因组维护领域的经验丰富和新手调查人员。

细胞不断暴露在 DNA 损伤的内源和外源, 威胁着基因组的完整性。DDR 是信号通路的集合, 用于检测、信号和修复 DNA 病变以维持基因组的稳定性。在 DNA 双链断裂 (DSBs), DDR 主要发生在两个互补平台上: γ H2A. X 标记染色质和切除的单链 DNA (ssDNA) 区域, 通常涂有 ssDNA 绑定的复杂 RPA1,2。
用胸苷模拟 5-溴-2 ' 脱氧尿苷 (BrdU) 或 bisbenzimide 乙醇三盐酸盐 (BBET, 赫斯特 33342) 对细胞进行预敏化的紫外激光微照射, 产生 dna 损伤的混合体, 包括单链断裂 (办学团体) 和 DSBs, 它在染色质和 ssDNA 平台上引出局部 DDR 3, 4.以前的研究表明, 在争端解决的这两个截然不同的平台上, 对基因组维护因素的招募可以用高能紫外线 (335-365 nm) 和如果2、5的组合来区分。配备此类激光器的显微镜成本高昂, 因为它们需要高能激光和专用紫外线--发射目标, 使它们远低于激光扫描共聚焦显微镜与405纳米激光的普及程度。线.在微辐照部位进行蛋白质的招募和交换的研究, 也被用来描述基因组维持因子的动态行为所需要的费力的人工图像分析所排除。
在这里, 我们表明, 对 BrdU 或 BBET 核酸染色的细胞进行预敏化, 然后用 405 nm 激光在共焦显微镜上进行微辐照, 可以监测 DNA 损伤基因组维持因子动态。使用伽玛 H2A X 或 RPA 复合子单元作为平台标记, 加上 z 堆叠, 以获得更大的景深和反褶积以提高分辨率, 使实验者能够区分当地招募到 DSBs 的因素, 从传播到早期病灶周围的大染色质域。这种分分类根据不同的核内隔间, 有助于改善 uncharacterized 蛋白的潜在作用, 招募到微辐照的网站。此外, 我们提供方便的协议和优化的管道, 以快速分析基因组维护因素的动态, 使用开放源码的软件斐济 (一个 ImageJ 分发)6,7,8。这些对目前微辐照方法的改进使对复员方案的研究在几乎任何实验室环境中都可能发生。

<table><tbody><tr><td>Olympus FLUOVIEW FV3000 resonant laser scanning confocal microscope</td><td>Olympus</td><td></td><td></td></tr><tr><td>FluoView FV3000 software (version 2.1)</td><td>Olympus</td><td></td><td></td></tr><tr><td>405 nm laser</td><td>Coherent</td><td></td><td>Radiation source</td></tr><tr><td>Microscope incubator AIO-UNO-OLYUS-1</td><td>Okolab</td><td>OKO-UNO-1</td><td></td></tr><tr><td>60X /1.4 Objective</td><td>Olympus</td><td></td><td>Oil immersion objective</td></tr><tr><td>8-well culture slides on coverglass (X-well)</td><td>Sarstedt</td><td>94.6190.802</td><td></td></tr><tr><td>U2OS osteosarcoma human cell line</td><td>ATCC</td><td>HTB-96</td><td>Stable cell lines&amp;nbsp;</td></tr><tr><td>4&amp;rsquo;,6-Diamidino-2-Phenylindole (DAPI)</td><td>ThermoFisher</td><td>D1306</td><td>Caution toxic</td></tr><tr><td>5-bromo-2&amp;prime;-deoxyuridine&amp;nbsp;(BrdU)&amp;nbsp;</td><td>Sigma-Aldrich</td><td>59-14-3</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich</td><td>30525-89-4</td><td>Caution toxic</td></tr><tr><td>Bisbenzimide H 33342 (Hoechst 33342)</td><td>ThermoFisher</td><td>62249</td><td></td></tr><tr><td>Bovine serum albumin (BSA)</td><td>ThermoFisher</td><td>BP1600-100</td><td></td></tr><tr><td>Trypsin EDTA 0.1%&amp;nbsp;</td><td>ThermoFisher</td><td>15400-054</td><td></td></tr><tr><td>Triton X-100&amp;nbsp;</td><td>BioShop</td><td>TRX777.100</td><td></td></tr><tr><td>Tween-20&amp;nbsp;</td><td>ThermoFisher</td><td>BP337-500</td><td></td></tr><tr><td>Sucrose&amp;nbsp;</td><td>BioShop</td><td>SUC507</td><td></td></tr><tr><td>JetPRIME transfection reagent&amp;nbsp;</td><td>Polyplus</td><td>114-07</td><td></td></tr><tr><td>ProLong Diamond Antifade mountant with DAPI&amp;nbsp;</td><td>ThermoFisher</td><td>P36962</td><td></td></tr><tr><td>DMEM 1X, + phenol red</td><td>Wisent&amp;nbsp;</td><td>319-005-CL</td><td></td></tr><tr><td>DMEM 1X, - phenol red</td><td>Wisent</td><td>319-051-CL</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Wisent&amp;nbsp;</td><td>080-150</td><td></td></tr><tr><td>Mouse anti-RPA32 antibody</td><td>Abcam</td><td>ab2175</td><td>1:500 for IF</td></tr><tr><td>Rabbit anti-&amp;gamma;-H2A.X antibody</td><td>Cell Signaling&amp;nbsp;</td><td>9718S</td><td>1:500 for IF</td></tr><tr><td>Rabbit Anti-53BP1 antibody</td><td>Cell Signaling&amp;nbsp;</td><td>4937S</td><td>1:500 for IF</td></tr><tr><td>Rabbit Anti-PRP19 antibody</td><td>Abcam</td><td>ab27692</td><td>1:500 for IF</td></tr><tr><td>Goat anti-rabbit IgG Alexa Fluor 647</td><td>Cell Signaling&amp;nbsp;</td><td>4414S</td><td>1:250 for IF</td></tr><tr><td>Goat anti-mouse&amp;nbsp; IgG Alexa Fluor 488&amp;nbsp;</td><td>Cell Signaling&amp;nbsp;</td><td>4408S</td><td>1:250 for IF</td></tr><tr><td>Fiji image analysis open-source software&amp;nbsp;</td><td></td><td></td><td>https://fiji.sc</td></tr><tr><td>1918-K Power meter&amp;nbsp; with a 918D-SL-0D3 sensor</td><td>Newport</td><td></td><td></td></tr></tbody></table>

investigation of protein recruitment to dna lesions using 405 nm laser micro-irradiation

dna 损伤反应 (DDR) 使用过多的蛋白质来检测、信号和修复 dna 损伤。描述这种反应对于了解基因组维护机制至关重要。由于在病变中的蛋白质的招募和交换是高度动态的, 他们的研究需要有能力以快速和空间分隔的方式产生 DNA 损伤。在这里, 我们描述的程序, 以当地诱发 DNA 损伤的人类细胞使用一个常用的激光扫描共聚焦显微镜配备405纳米激光线。基因组维持因子在激光条纹上的积累可以用免疫荧光 (IF) 或实时使用荧光记者标记的蛋白质来评估。使用磷酸化组蛋白 H2A。x (γ H2A x) 和复制蛋白 A (RPA) 作为标记, 该方法提供了足够的分辨率, 以区分当地征聘的因素, 从传播到相邻的染色质。我们进一步提供基于 ImageJ 的脚本, 以有效地监测 DNA 损伤部位蛋白质孵育由的动力学。这些改进大大简化了对复员方案动力学的研究。

在微辐照后, 根据基因组维护蛋白1、12的性质, 允许细胞在特定时间段内恢复。DSBs 将被核酸, 最广泛的过程中的 S 和 G2 阶段的细胞周期, 以创建一个有限的区域 ssDNA, 这是迅速覆盖的爱国军和其他基因组维护因素9。这个 ssDNA 地区四周都是染色质, 在复员方案期间进行了广泛的修改, 以管制其他复员方案因素的招募和交换13。可通过 (图 1A) 监视微辐照区域的蛋白质招募。通常, 染色质相关因素 (例如、γ H2A X、53BP1、等) 在较 ssDNA 的蛋白质的早期 (1–5) 时间点上可以检测到, 因为核酸酶介导的争端终止的切除需要生成 RPA-ssDNA 平台。(≥10分钟)。为了便于图像分析和发布, 我们设计了一个斐济脚本 (Outliner)10 , 它使用 DNA 染料相关通道 (例如、DAPI) 自动勾勒出细胞核, 从而更容易区分微辐射条纹和单个细胞通过在保存核定义的同时保留最终图像的言之无物 DNA 染色 (图 1B)。
伽玛 H2A X 和爱国军复合亚基的功能作为标记的染色质-和 ssDNA 相关的因素, 分别。爱国军-ssDNA 显示为有斑点的病灶周围的大荧光染色质条纹装饰的γ-H2A. X 抗体 (图 1C)。这些标记的协同定位可以用来精确定义基因组维持因子在 DNA 损伤中的位置。例如, 53BP1 是一种染色质相关蛋白, 它控制着争端处理的修复路径选择, 与γ H2A 本地化良好. X 信号13。相反, PRP19, 一个 E3 泛素连接酶, 在 RPA ssDNA 的作用, 以促进 ATR 激活和 DNA 修复被招募为有点的焦点, 密切匹配 RPA32 分布 14, 15, 16, 17(图 1D)。值得注意的是, 一些因素也可能被招募到与最初病变相邻的染色质上, 但不会扩散到 DSBs 周围的大范围, 如γ-H2A. X 和53BP1。在这种情况下, 这些因素会出现有点点的焦点, 但不会完全与 ssDNA 结合的蛋白质的本地化。
对于活体细胞成像, 用荧光标记标记的细胞可以进行微辐照和实时成像, 以获得高度详细的招聘动力学 (图 2AC)。可以同时观察多种不同的蛋白质, 条件是它们的荧光标签可以相互分离。为了便于分析时间序列, 我们创建了两个斐济脚本, 允许用户制作包含时间序列信息的电影文件 (电影制作者, 参见图 2C的电影作为在线补充材料) 并量化蛋白质在微辐照站点 (损坏分析器) 的招聘10。损坏分析器脚本不是通常需要描述蛋白质行为的耗时的手动图像精选, 而是通过简化的步骤来指导用户, 该过程监视微辐照站点上的信号浓缩, 同时自动在实验的所有时间点对细胞运动、图像漂移、背景信号和漂白进行校正。然后, 生成的数据可以很容易地编译到电子表格应用程序中, 并根据需要绘制 (图 2D)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57410/57410fig1v2.jpg"> 图 1:基因组维持因子在微辐照诱发的 ssDNA 平台上的定位.(A) 在预敏细胞中的激光微辐照导致 DNA 双链断裂的产生, 这是由内核酸和外源切除产生 ssDNA。ssDNA 子室可以被可视为有点的焦点使用抗体反对复制蛋白 A 或其他 ssDNA 局部因素。与此相反, 扩散到大染色质域的蛋白质或修饰在微辐照部位出现为大条纹, 类似于以磷酸化组蛋白变异 H2A 为靶点的抗体。x (γ H2A x)。(B) 预敏与 BBET 或 BrdU 的细胞微辐照, 预提取, 固定, 并染色所述蛋白。12位图像是在1X 缩放时收集的, 使用 Outliner 斐济脚本进行处理。(C) BBET 或 BrdU 预敏细胞的 Z 叠加允许解析 ssDNA 和染色质相关因子 (显示的最大强度 Z 投影)。(D) 使用此方法, 53BP1 和 PRP19 可以分别归类为染色质和 ssDNA 相关的基因组维持因子。刻度条 = 10 µm;D 中的图像的比例都相同。<a href="//cloudflare.jove.com/files/ftp_upload/57410/57410fig1v2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57410/57410fig2.jpg"> 图 2: 对爱国军复杂招募到 DNA 损伤部位的实时成像.(A) 稳定或瞬态地转染由荧光记者标记的候选蛋白的质粒, 其次是对被转染细胞进行微辐照, 从而能够实时监测 DNA 损伤的蛋白质招募情况。(B) 细胞用 pDEST47-RPA32-GFP 质粒和裂解24小时后转染。蛋白表达经免疫印迹法证实。(C) 用 pDEST-SFB EGFP 或 pDEST47 RPA32-GFP 质粒对 BrdU 进行预敏化的细胞进行微辐照, 并在每分钟照射后1X 变焦时成像12位。时间在照射后几分钟内。00:00 时间点图像是在微辐照之前拍摄的。电影文件是使用电影制作者斐济脚本和关键帧显示。刻度条 = 10 µm. (D) 使用 "损坏分析器" 斐济脚本对微辐照条纹的 RPA32-GFP 进行定量监测。输出数据是使用 Microsoft Excel 绘制的。黑线是 RPA32-GFP 在微辐照部位相对于预辐照信号的平均富集褶皱, 误差条代表15独立微辐照细胞平均值的标准误差。<a href="//cloudflare.jove.com/files/ftp_upload/57410/57410fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Movie 1" src="/files/ftp_upload/57410/57410mov1.jpg"> 影片 1: SFB-GFP 表达细胞的微辐照时间间隔.细胞瞬时转染的 pDEST SFB GFP 质粒是预敏24小时与 BrdU, 微辐照, 并成像每分钟一次29分钟.<a href="//cloudflare.jove.com/files/ftp_upload/57410/57410_Movie_1.avi" target="_blank">请单击此处下载此影片.</a>
<img alt="Movie 2" src="/files/ftp_upload/57410/57410mov2.jpg"> 影片 2: RPA32-GFP 表达细胞的微辐照时间间隔.细胞瞬时转染的 pDEST47 RPA32-GFP 质粒是预敏24小时与 BrdU, 微辐照, 并成像每分钟一次29分钟.<a href="//cloudflare.jove.com/files/ftp_upload/57410/57410_Movie_2.avi" target="_blank">请单击此处下载此影片.</a>

Watch this Scientific Journal Video about Investigation of Protein Recruitment to DNA Lesions Using 405 Nm Laser Micro-irradiation at JoVE.com

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

研究 DNA 损伤修复动力学需要一个系统来诱发定义的亚核区域的病变。我们描述了一个方法来创建本地化的双绞合, 使用激光扫描共聚焦显微镜配备405纳米激光, 并提供自动化程序, 以量化的修复因素在这些病变的动态。

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 ...

investigation-protein-recruitment-to-dna-lesions-using-405-nm-laser

Nanyang Technological University

Research

JoVE Journal

Genetics

9.4K 次观看.  Université de Sherbrooke. 研究 DNA 损伤修复动力学需要一个系统来诱发定义的亚核区域的病变。我们描述了一个方法来创建本地化的双绞合, 使用激光扫描共聚焦显微镜配备405纳米激光, 并提供自动化程序, 以量化的修复因素在这些病变的动态。

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

RNA Interference-based Investigation of the Function of Heat Shock Protein 27 during Corneal Epithelial Wound Healing

Small interfering RNA (siRNA) is among the most widely used RNA interference methods for the short-term silencing of protein-coding genes. siRNA is a synthetic RNA duplex created to specifically target a mRNA transcript to induce its degradation and it has been used to identify novel pathways in various cellular processes. Few reports exist regarding the role of phosphorylated heat shock protein 27 (HSP27) in corneal epithelial wound healing. Herein, cultured human corneal epithelial cells were divided into a scrambled control-siRNA transfected group and a HSP27-specific siRNA-transfected group. Scratch-induced directional wounding assays, and western blotting, and flow cytometry were then performed. We conclude that HSP27 has roles in corneal epithelial wound healing that may involve epithelial cell apoptosis and migration. Here, step-by-step descriptions of sample preparation and the study protocol are provided.

Herein, we present a protocol to use heat shock protein 27 (HSP27)-specific small interfering RNA to assess the function of HSP27 during corneal epithelial wound healing. RNA interference is the best method for effectively knocking-down gene expression to investigate protein function in various cell types.

角膜上皮伤口愈合过程中的热休克蛋白27的功能为基础的RNA干扰研究

Small interfering RNA (siRNA) is among the most widely used RNA interference methods for the short-term silencing of protein-coding genes. siRNA is a ...

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, ...

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

Protocols for C-Brick DNA Standard Assembly Using Cpf1

CRISPR-associated protein Cpf1 cleaves double-stranded DNA under the guidance of CRISPR RNA (crRNA), generating sticky ends. Because of this characteristic, Cpf1 has been used for the establishment of a DNA assembly standard called C-Brick, which has the advantage of long recognition sites and short scars. On a standard C-Brick vector, there are four Cpf1 recognition sites &#8211; the prefix (T1 and T2 sites) and the suffix (T3 and T4 sites) &#8211; flanking biological DNA parts. The cleavage of T2 and T3 sites produces complementary sticky ends, which allow for the assembly of DNA parts with T2 and T3 sites. Meanwhile, a short &#34;GGATCC&#34; scar is generated between parts after assembly. As the newly formed plasmid once again contains the four Cpf1 cleavage sites, the method allows for the iterative assembly of DNA parts, which is similar to those of BioBrick and BglBrick standards. A procedure outlining the use of the C-Brick standard to assemble DNA parts is described here. The C-Brick standard can be widely used by scientists, graduate and undergraduate students, and even amateurs.

CRISPR-associated protein Cpf1 can be guided by a specially designed CRISPR RNA (crRNA) to cleave double-stranded DNA at desired sites, generating sticky ends. Based on this characteristic, a DNA assembly standard (C-Brick) was established, and a protocol detailing its use is described here.

使用Cpf1的C-Brick DNA标准装配的方案

CRISPR-associated protein Cpf1 cleaves double-stranded DNA under the guidance of CRISPR RNA (crRNA), generating sticky ends. Because of this ...

Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae

Genome-wide mapping of protein-DNA interactions is critical for understanding gene regulation, chromatin remodeling, and other chromatin-resident processes. Formaldehyde crosslinking followed by chromatin immunoprecipitation and high-throughput sequencing (X-ChIP-seq) has been used to gain many valuable insights into genome biology. However, X-ChIP-seq has notable limitations linked to crosslinking and sonication. Native ChIP avoids these drawbacks by omitting crosslinking, but often results in poor recovery of chromatin-bound proteins. In addition, all ChIP-based methods are subject to antibody quality considerations. Enzymatic methods for mapping protein-DNA interactions, which involve fusion of a protein of interest to a DNA-modifying enzyme, have also been used to map protein-DNA interactions. We recently combined one such method, chromatin endogenous cleavage (ChEC), with high-throughput sequencing as ChEC-seq. ChEC-seq relies on fusion of a chromatin-associated protein of interest to micrococcal nuclease (MNase) to generate targeted DNA cleavage in the presence of calcium in living cells. ChEC-seq is not based on immunoprecipitation and so circumvents potential concerns with crosslinking, sonication, chromatin solubilization, and antibody quality while providing high resolution mapping with minimal background signal. We envision that ChEC-seq will be a powerful counterpart to ChIP, providing an independent means by which to both validate ChIP-seq findings and discover new insights into genomic regulation.

Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae

We describe chromatin endogenous cleavage coupled with high-throughput sequencing (ChEC-seq), a chromatin immunoprecipitation (ChIP)-orthogonal method for mapping protein binding sites genome-wide with micrococcal nuclease (MNase) fusion proteins.

与ChEC-seq的蛋白质-DNA相互作用的全基因组映射<em&gt;酿酒酵母（Saccharomyces cerevisiae）</em

Genome-wide mapping of protein-DNA interactions is critical for understanding gene regulation, chromatin remodeling, and other chromatin-resident ...

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.

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.

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 ...

Detection and Visualization of DNA Damage-induced Protein Complexes in Suspension Cell Cultures Using the Proximity Ligation Assay

The DNA damage response orchestrates the repair of DNA lesions that occur spontaneously, are caused by genotoxic stress, or appear in the context of programmed DNA breaks in lymphocytes. The Ataxia-Telangiectasia Mutated kinase (ATM), ATM- and Rad3-Related kinase (ATR) and the catalytic subunit of DNA-dependent Protein Kinase (DNA-PKcs) are among the first that are activated upon induction of DNA damage, and are central regulators of a network that controls DNA repair, apoptosis and cell survival. As part of a tumor-suppressive pathway, ATM and ATR activate p53 through phosphorylation, thereby regulating the transcriptional activity of p53. DNA damage also results in the formation of so-called ionizing radiation-induced foci (IRIF) that represent complexes of DNA damage sensor and repair proteins that accumulate at the sites of DNA damage, which are visualized by fluorescence microscopy. Co-localization of proteins in IRIFs, however, does not necessarily imply direct protein-protein interactions, as the resolution of fluorescence microscopy is limited. 
In situ Proximity Ligation Assay (PLA) is a novel technique that allows the direct visualization of protein-protein interactions in cells and tissues with unprecedented specificity and sensitivity. This technique is based on the spatial proximity of specific antibodies binding to the proteins of interest. When the interrogated proteins are within ~40 nm an amplification reaction is triggered by oligonucleotides that are conjugated to the antibodies, and the amplification product is visualized by fluorescent labeling, yielding a signal that corresponds to the subcellular location of the interacting proteins. Using the established functional interaction between ATM and p53 as an example, it is demonstrated here how PLA can be used in suspension cell cultures to study the direct interactions between proteins that are integral parts of the DNA damage response.

Here, it is demonstrated how the in situ Proximity Ligation Assay (PLA) can be used to detect and visualize the direct protein-protein interactions between ATM and p53 in suspension cell cultures exposed to genotoxic stress.

检测和可视化DNA损伤诱导的蛋白质复合物悬浮细胞培养使用邻近连接测定

The DNA damage response orchestrates the repair of DNA lesions that occur spontaneously, are caused by genotoxic stress, or appear in the context of ...

生物化学

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 ...