Real-time imaging of DNA damage in yeast cells using ultra-short near-infrared pulsed laser irradiation.
Real-time imaging of DNA damage in yeast cells using ultra-short near-infrared pulsed laser irradiation.
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DOI：
10.1371/journal.pone.0113325
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Kearsey SE
中科院分区：
文献类型：
--
作者：
Guarino E;Cojoc G;García-Ulloa A;Tolić IM;Kearsey SE
对酵母细胞核修复位点修复和检查点蛋白积累的分析通常使用化学试剂、电离辐射或诱导核酸内切酶来造成局部损伤。除了这些方法之外，在哺乳动物细胞中进行的类似研究还使用了激光照射，其优点是在特定的核区域和精确的时间造成损伤，这使得可以对 DNA 损伤位点的蛋白质积累进行准确的动力学分析。我们在这里表明，使用近红外激光照射的短脉冲通过多光子吸收对酵母细胞核的亚核区域造成 DNA 损伤是可行的。与荧光标记的蛋白质结合使用，可以在损伤诱导的几秒钟内对损伤部位的蛋白质积累进行定量分析。 PCNA 在损伤部位迅速积累，损伤后约 50 秒出现最大积累，然后水平在照射后 200-1000 秒内线性下降。 RPA 以较慢的动力学积累，因此在照射后 60 秒内几乎检测不到任何积累，并且其水平随后在接下来的 900 秒内线性增加，此后损伤部位的水平大致恒定（最多约 2700 秒）。这种方法补充了现有的方法，可以分析关键损伤传感器和损伤开始后几秒钟内发生的染色质修饰变化。
Analysis of accumulation of repair and checkpoint proteins at repair sites in yeast nuclei has conventionally used chemical agents, ionizing radiation or induction of endonucleases to inflict localized damage. In addition to these methods, similar studies in mammalian cells have used laser irradiation, which has the advantage that damage is inflicted at a specific nuclear region and at a precise time, and this allows accurate kinetic analysis of protein accumulation at DNA damage sites. We show here that it is feasible to use short pulses of near-infrared laser irradiation to inflict DNA damage in subnuclear regions of yeast nuclei by multiphoton absorption. In conjunction with use of fluorescently-tagged proteins, this allows quantitative analysis of protein accumulation at damage sites within seconds of damage induction. PCNA accumulated at damage sites rapidly, such that maximum accumulation was seen approximately 50 s after damage, then levels declined linearly over 200–1000 s after irradiation. RPA accumulated with slower kinetics such that hardly any accumulation was detected within 60 s of irradiation, and levels subsequently increased linearly over the next 900 s, after which levels were approximately constant (up to ca. 2700 s) at the damage site. This approach complements existing methodologies to allow analysis of key damage sensors and chromatin modification changes occurring within seconds of damage inception.
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影响因子：
4.8
作者：
Kim, JS;Krasieva, TB;Yokomori, K
通讯作者：
Yokomori, K
影响因子：
64.5
作者：
Lambert, S;Watson, A;Carr, AM
通讯作者：
Carr, AM
DOI：
10.1016/0027-5107(91)90177-p
发表时间：
1991-09-01
期刊：
MUTATION RESEARCH
影响因子：
--
作者：
LEHMANN, AR;CARR, AM;MURRAY, JM
通讯作者：
MURRAY, JM
DOI：
10.1073/pnas.0406048101
发表时间：
2004-09-21
影响因子：
11.1
作者：
Lan, L;Nakajima, S;Yasui, A
通讯作者：
Yasui, A
影响因子：
3.7
作者：
Ferrando-May E;Tomas M;Blumhardt P;Stöckl M;Fuchs M;Leitenstorfer A
通讯作者：
Leitenstorfer A