Giannattasio Michele, Lazzaro Federico, Longhese Maria Pia, Plevani Paolo, Muzi-Falconi Marco
Dipartimento di Scienze Biomolecolari e Biotecnologie, Università degli Studi di Milano, Milano, Italy.
EMBO J. 2004 Jan 28;23(2):429-38. doi: 10.1038/sj.emboj.7600051. Epub 2004 Jan 15.
The mechanisms used by checkpoints to identify DNA lesions are poorly understood and may involve the function of repair proteins. Looking for mutants specifically defective in activating the checkpoint following UV lesions, but proficient in the response to methyl methane sulfonate and double-strand breaks, we isolated cdu1-1, which is allelic to RAD14, the homolog of human XPA, involved in lesion recognition during nucleotide excision repair (NER). Rad14 was also isolated as a partner of the Ddc1 checkpoint protein in a two-hybrid screening, and physical interaction was proven by co-immunoprecipitation. We show that lesion recognition is not sufficient for checkpoint activation, but processing, carried out by repair factors, is required for recruiting checkpoint proteins to damaged DNA. Mutations affecting the core NER machinery abolish G1 and G2 checkpoint responses to UV, preventing activation of the Mec1 kinase and its binding to chromosomes. Conversely, elimination of transcription-coupled or global genome repair alone does not affect checkpoints, suggesting a possible interpretation for the heterogeneity in cancer susceptibility observed in different NER syndrome patients.
关卡用于识别DNA损伤的机制尚不清楚，可能涉及修复蛋白的功能。为了寻找在紫外线损伤后激活关卡存在特异性缺陷，但对甲基磺酸甲酯和双链断裂反应正常的突变体，我们分离出了cdu1-1，它与RAD14等位，RAD14是人类XPA的同源物，参与核苷酸切除修复（NER）过程中的损伤识别。在双杂交筛选中，Rad14也被分离为Ddc1关卡蛋白的一个伴侣，并且通过免疫共沉淀证明了两者之间的物理相互作用。我们发现损伤识别不足以激活关卡，但是由修复因子进行的处理对于将关卡蛋白募集到受损DNA上是必需的。影响核心NER机制的突变消除了G1和G2关卡对紫外线的反应，阻止了Mec1激酶的激活及其与染色体的结合。相反，单独消除转录偶联修复或全基因组修复并不影响关卡，这为在不同NER综合征患者中观察到的癌症易感性异质性提供了一种可能的解释。
