Department of Microbiology and Immunology, University of Saskatchewan, Saskatoon, S7N 5E5, Canada.
Mol Biol Cell. 2011 Jul 1;22(13):2373-83. doi: 10.1091/mbc.E10-12-0938. Epub 2011 May 5.
In response to DNA damage such as from UV irradiation, mammalian Y-family translesion synthesis (TLS) polymerases Polη and Rev1 colocalize with proliferating cell nuclear antigen at nuclear foci, presumably representing stalled replication sites. However, it is unclear whether the localization of one polymerase is dependent on another. Furthermore, there is no report on the in vivo characterization of the Rev3 catalytic subunit of the B-family TLS polymerase Polζ. Here we describe the detection of endogenous human Polη, Rev1, and Rev3 by immunocytochemistry using existing or newly created antibodies, as well as various means of inhibiting their expression, which allows us to examine the dynamics of endogenous TLS polymerases in response to UV irradiation. It is found that Rev1 and Polη are independently recruited to the nuclear foci, whereas the Rev3 nuclear focus formation requires Rev1 but not Polη. In contrast, neither Rev1 nor Polη recruitment requires Rev3. To further support these conclusions, we find that simultaneous suppression of Polη and Rev3 results in an additive cellular sensitivity to UV irradiation. These observations suggest a cooperative and sequential assembly of TLS polymerases in response to DNA damage. They also support and extend the current polymerase switch model.
针对诸如来自 UV 照射的 DNA 损伤，哺乳动 物 Y 家族跨损伤合成（TLS）聚合酶 Polη 和 Rev1 与增殖细胞核抗原在核斑中共定位，推测代表停滞的复制位点。然而，尚不清楚一种聚合酶的定位是否依赖于另一种聚合酶。此外，尚无关于 B 家族 TLS 聚合酶 Polζ 的 Rev3 催化亚基的体内特征的报道。在这里，我们使用现有的或新创建的抗体通过免疫细胞化学检测内源性人 Polη、Rev1 和 Rev3，以及各种抑制其表达的方法，这使我们能够检查内源性 TLS 聚合酶对 UV 照射的反应动力学。结果发现，Rev1 和 Polη 独立地被募集到核斑中，而 Rev3 的核斑形成需要 Rev1 但不需要 Polη。相比之下，Rev1 和 Polη 的募集都不需要 Rev3。为了进一步支持这些结论，我们发现同时抑制 Polη 和 Rev3 会导致细胞对 UV 照射的敏感性增加。这些观察结果表明 TLS 聚合酶在响应 DNA 损伤时会发生协作和顺序组装。它们还支持和扩展了当前的聚合酶切换模型。
