Investigating the role of XPB helicase in DNA nucleotide excision repair

 描述(申请人提供)：着色性干皮病(XP)B组(XPB)蛋白是转录和核苷酸切除修复(NER)所需的DNA解旋酶，NER是去除各种DNA损伤的主要DNA修复途径。XPB基因突变与皮肤癌易感性、过早衰老和神经变性有关。为了解决有关NER的两个长期存在的问题：XPB如何在损伤部位启动dsDNA解离以及损伤DNA的打开如何与双重切开相协调，我们计划实现以下三个具体目标：目的1：剖析XPB在NER中解离DNA的机制。我们将在含有(6-4)嘧啶-嘧啶二聚体的气泡与dsDNA和DNA的复合体中测定人XPB的晶体结构。通过小角X射线散射(SAXS)实验，进一步分析了XPB与DNA的动态相互作用和功能构象。这些结构模型将通过体外生化分析的选择性突变和基于细胞的成像实验来验证，以测试XPB在活细胞中NER过程中的作用。目的：阐明XPB-XPF相互作用通过内切酶ERCC1-XPF调节5‘-切割。XPB的Ser751位残基的磷酸化抑制了ERCC1-XPF对损伤DNA的5‘-切割，因此XPB起到了调节双重切割的作用。我们已经纯化了人XPB C-末端一半与XPF的N-末端一半的复合体，用于结构和功能分析。一种新的假说是如何通过XPB的Ser751位的磷酸化来抑制XPF的5‘-切割，将通过体外生化分析的选择性突变和活细胞损伤切割的细胞成像实验来检验。目的：阐明致病基因XPB突变引起的结构和功能变化。目前文献中仅发现3种氨基酸替代突变：F99S突变引起XP，T119P突变引起毛发硫代营养不良(TTD)，XP11BE突变引起XP/Cockayne综合征(CS)综合征。XP11BE是一种移码突变，它改变了XPB C末端的最后42个残基。已知XP11BE突变取消了XPB与XPF相互作用的基序，导致XP表型。我们的初步结果表明，与野生型XPB相比，最后42个氨基酸残基的变化降低了XPB突变体的溶解性，从而导致TFIIH的细胞水平有限，进而导致CS表型的霍尔标志-恢复RNA合成减少。关于邻近突变F99S和T119P为什么会导致不同的表型，目前还知之甚少。为了从化学和原子水平检测这两种突变，我们将制备含有F99S和T119P突变的XPB重组蛋白进行结构和功能分析。这些结果将对了解这些突变的致病机制非常重要，并将具有重大的医学意义。

 DESCRIPTION (provided by applicant): Xeroderma pigmentosum (XP) group B (XPB) protein is a DNA helicase required for both transcription and nucleotide excision repair (NER), a major DNA repair pathway that removes a variety of DNA lesions. Mutations in the XPB gene are associated with skin cancer predisposition, premature aging, and neurodegeneration. In order to solve two long-standing problems concerning NER: how XPB initiates dsDNA unwinding at the damage site and how the opening of damaged DNA is coordinated with the dual incision, we plan to achieve the following three specific aims: Aim1: To dissect the mechanism of DNA unwinding by XPB during NER. We will determine the crystal structures of human XPB in complex with dsDNA and DNA with a bubble containing a lesion such as (6-4) pyrimidine-pyrimidone dimer. The dynamic XPB-DNA interactions and functional conformations of XPB will be further analyzed by small angle X-ray scattering (SAXS) experiments. These structural models will be validated by selective mutagenesis for in vitro biochemical assays and cell-based imaging experiments to test the role of XPB during NER in living cells. Aim2: To illustrate the XPB-XPF interaction that regulates the 5'-incison by endonuclease ERCC1-XPF. XPB plays a role in regulating the dual incision since phosphorylation at residue Ser751 of XPB inhibits the 5'- incison of the damaged DNA by ERCC1-XPF. We have purified a complex of human XPB C-terminal half with the N-terminal half of XPF for structural and functional analyses. A novel hypothesis how phosphorylation at Ser751 of XPB inhibits 5'-incision by XPF will be tested by selective mutagenesis for in vitro biochemical assays and cell-based imaging experiments for damage incision in the living cells. Aim3: To illustrate the structural and functional changes induced by disease-causing xpb mutations. There are only three amino acid substitution mutations identified so far in the literatures: F99S mutation causes XP, T119P mutation causes Trichothiodystrophy (TTD), and XP11BE mutation causes XP/Cockayne Syndrome (CS) complex. XP11BE is a frame-shift mutation that changes the last 42 residues at the C-terminus of XPB. It has been known that XP11BE mutation abolishes the interaction motif of XPB with XPF leading to XP phenotype. Our preliminary results indicate that change of the last 42 amino acid residues reduces the solubility of XPB mutant compared to the wild type XPB, therefore leading to limited cellular level of TFIIH which in turn causes reduced recovery RNA synthesis, the hall mark of CS phenotype. There is little information about why the adjacent mutations F99S and T119P cause different phenotypes. In order to examine these two mutations at chemical and atomic level, XPB recombinant proteins bearing F99S and T119P mutations will be prepared for structural and functional analysis. These results will be very important for understanding the pathogenic mechanism of these mutations and will have significant medical implications.