DNA IR-damage and cellular response via ATR

In fission yeast, the rad3 gene product plays a critical role in sensing DNA structure defects and activating damage response pathways. A structural homologue of rad3 in humans has been identified based on sequence similarity in the protein kinase domain. ATR (for Ataxia Telangiectasia and Rad3-related) is considered the mammalian counterpart of yeast rad3, Mec1p, and fruit fly Mei-41, proteins involved in DNA damage responses. The ATR protein is a member of the phosphoinositide 3-kinase related kinase family and plays an important role in UV-induced DNA damage checkpoint response and its role as a signal transducer in cell cycle checkpoint has been well established. Even though it is currently unclear whether ATR functions as a damage sensor, recent evidence shows that ATR may function as an initial sensor in the DNA damage checkpoint response. Moreover, it has been found that ATR is a DNA-binding protein with higher affinity to UV-damaged than undamaged DNA. In addition, damaged DNA stimulates the kinase activity of ATR to a significantly higher level than undamaged DNA. ATR is structurally related to ATM (for Ataxia Telangiectasia Mutated) as well as the yeast PIK family members Mec1p and Rad3. Mec1p and Rad3 participate in checkpoint pathways induced by DNA replication blocks, DNA strand breaks, and other chromosomal abnormalities, which implies that ATR performs similar functions in mammalian cells. Reports have demonstrated that overexpression of a catalytically inactive version of ATR (ATRki) in human fibroblasts caused hypersensitivity to gamma-radiation and hydroxyurea and abrogation of the radiation-induced G2 checkpoint. The checkpoint defects observed in ATR-overexpressing cells resemble those found in AT cells. Additionally, ATR functions as an upstream regulator of p53 phosphorylation in DNA-damaged cells. ATR phosphorylates p53 at both Ser-15 and Ser-37 in vitro, suggesting that ATR is directly involved in the modification of p53 in DNA-damaged cells. Recent reports concerning ATM, suggest that ATR and ATM play both overlapping and independent roles in the phosphorylation of p53 during cellular exposure to genotoxic stress. Atr is localized to the nuclei of primary spermatocytes, cells that are undergoing meiosis I. It has been demonstrated that both Atr and Atm proteins have associated protein kinase activity, consistent with their primary structures. Additionally, Atr and Atm show specific association with chromosomes in cells that are in early meiosis I as demonstrated by antibody localization on surface-spread spermatocytes. Both the Atr and Atm proteins are present at pairing forks in meiotic prophase as chromosomes synapse; however, they do not colocalize, instead they occupy complementary positions: Atr localizes along unsynapsed chromosome axes and Atm interacts with synapsed axes. Meanwhile ATM is activated by damage-induced rapid intermolecular autophosphorylation prior relocalization to sites of DNA breaks, ATR activation seems to require single-stranded DNA (ssDNA) coated with replication protein A. The recruitment of ATR to damage sites appears to be mediated by an ATR-interacting protein that forms a stable complex with the vast majority of ATR in human cells.

在裂殖酵母中，rad3基因产物在感知DNA结构缺陷和激活损伤响应通路中发挥着至关重要的作用。基于蛋白质激酶结构域的序列相似性，已在人类中鉴定出rad3的结构同源物。ATR（Ataxia Telangiectasia and Rad3-related，即共济失调性毛细血管扩张和Rad3相关蛋白）被认为是酵母rad3、Mec1p以及果蝇Mei-41蛋白在哺乳动物中的对应物，这些蛋白均参与DNA损伤响应。ATR蛋白是磷脂酰肌醇3激酶相关激酶家族的成员，在紫外线诱导的DNA损伤检查点响应中发挥着重要作用，其作为细胞周期检查点信号转导者的角色已得到充分确认。尽管目前尚不清楚ATR是否作为损伤传感器发挥作用，但最新证据表明，ATR可能在DNA损伤检查点响应中充当初始传感器。此外，研究发现ATR是一种DNA结合蛋白，与未受损DNA相比，对紫外线受损DNA的亲和力更高。此外，受损DNA刺激ATR激酶活性的程度显著高于未受损DNA。ATR在结构上与ATM（Ataxia Telangiectasia Mutated，即共济失调性毛细血管扩张突变蛋白）以及酵母PIK家族成员Mec1p和Rad3相关。Mec1p和Rad3参与由DNA复制阻塞、DNA单链断裂和其他染色体异常引发的检查点通路，这表明ATR在哺乳细胞中执行类似的功能。有报道显示，在人类成纤维细胞中过表达催化不活性的ATR（ATRki）版本会导致对γ射线和羟脲的超敏反应，并消除辐射诱导的G2检查点。在ATR过表达细胞中观察到的检查点缺陷与AT细胞中的缺陷相似。此外，ATR在DNA受损细胞中作为p53磷酸化的上游调节因子发挥作用。体外实验表明，ATR在Ser-15和Ser-37处磷酸化p53，表明ATR直接参与DNA受损细胞中p53的修饰。最近关于ATM的报告表明，在细胞暴露于致突变应激期间，ATR和ATM在p53磷酸化中既发挥重叠作用，也发挥独立作用。Atr定位于初级精母细胞的细胞核中，这些细胞正在进行减数第一次分裂。研究表明，Atr和Atm蛋白都具有与它们的初级结构一致的蛋白激酶活性。此外，Atr和Atm在与早期减数第一次分裂细胞的染色体特异性结合中表现出特定的相关性，这通过表面展开的精母细胞上的抗体定位得到证实。在减数分裂前期，Atr和Atm蛋白均存在于配对叉处，当染色体发生联会时；然而，它们并不共定位，而是占据互补的位置：Atr定位于未联会染色体的轴线上，而Atm与联会轴相互作用。与此同时，ATM在重新定位到DNA断裂部位之前，通过损伤诱导的快速分子内自磷酸化而被激活，而ATR的激活似乎需要被复制蛋白A覆盖的单链DNA（ssDNA）。ATR被招募到损伤位点似乎是通过一种与ATR相互作用的蛋白介导的，该蛋白与人体细胞中大部分ATR形成稳定的复合物。