Human damage-specific DNA-binding protein p48 - Characterization of XPE mutations and regulation following UV irradiation
Human damage-specific DNA-binding protein p48 - Characterization of XPE mutations and regulation following UV irradiation
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DOI：
10.1074/jbc.m000960200
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发表时间：
2000-07-14
影响因子：
4.8
通讯作者：
Linn, S
中科院分区：
文献类型：
--
作者：
Nichols, AF;Itoh, T;Linn, S
损伤特异性DNA结合（DDB）活性从HeLa细胞中纯化为异源二聚体（p127和p48），并且在着色性干皮病E组（XPE）患者的亚组（Ddb（-））细胞中不存在。每个亚基在昆虫细胞中过表达并纯化。对于HeLa异源二聚体特征的受损DNA带移，两者都必须存在。然而，含有在三个Ddb（-）XPE菌株中发现的突变的过表达的p48肽是无活性的，并且野生型p48恢复了来自第四个XPE Ddb（-）菌株GM 01389的提取物的DDB活性，其中DDB 2（p48）中的复合杂合突变导致一个等位基因的L350 P改变和另一个等位基因的Asn-349缺失。虽然这些结果表明，这些突变是每个负责DDB活性的损失，他们不影响核定位的p48。在正常的成纤维细胞中，4倍的p48 mRNA量增加，观察到紫外线照射后38小时，在48小时的p48蛋白和DDB活性类似的升高之前，这意味着p48限制DDB活性在体内。由于DNA修复在48小时前几乎完成，因此建议DDB的作用不同于DNA修复。
Damage-specific DNA binding (DDB) activity purifies from HeLa cells as a heterodimer (p127 and p48) and is absent from cells of a subset (Ddb(-)) of xeroderma pigmentosum Group E (XPE) patients. Each subunit was overexpressed in insect cells and purified. Both must be present for the damaged DNA band shift characteristic of the HeLa heterodimer. However, overexpressed p48 peptides containing the mutations found in three Ddb(-) XPE strains are inactive, and wild type p48 restores DDB activity to extracts from a fourth XPE Ddb(-) strain, GM01389, in which compound heterozygous mutations in DDB2 (p48) lead to a L350P change from one allele and a Asn-349 deletion from the other. Although these results indicate that these mutations are each responsible for the loss of DDB activity, they do not affect nuclear localization of p48. In normal fibroblasts, a 4-fold increase in p48 mRNA amount was observed 38 h after UV irradiation, preceding a similar elevation in p48 protein and DDB activity at 48 h, implying that p48 limits DDB activity in vivo. Because DNA repair is virtually complete before 48 h, a role for DDB other than DNA repair is suggested.