Lee Joon-Hwa, Park Chin-Ju, Arunkumar Alphonse I, Chazin Walter J, Choi Byong-Seok
Department of Chemistry and National Creative Research Initiative Center, Korea Advanced Institute of Science and Technology, 373-1, Guseong-dong, Yuseong-gu, Daejon 305-701, Korea.
Nucleic Acids Res. 2003 Aug 15;31(16):4747-54. doi: 10.1093/nar/gkg683.
In mammalian cells, nucleotide excision repair (NER) is the major pathway for the removal of bulky DNA adducts. Many of the key NER proteins are members of the XP family (XPA, XPB, etc.), which was named on the basis of its association with the disorder xerodoma pigmentosum. Human replication protein A (RPA), the ubiquitous single-stranded DNA-binding protein, is another of the essential proteins for NER. RPA stimulates the interaction of XPA with damaged DNA by forming an RPA-XPA complex on damaged DNA sites. Binding of RPA to the undamaged DNA strand is most important during NER, because XPA, which directs the excision nucleases XPG and XPF, must bind to the damaged strand. In this study, nuclear magnetic resonance (NMR) spectroscopy was used to assess the binding of the tandem high affinity DNA-binding domains, RPA-AB, and of the isolated domain RPA-A, to normal DNA and damaged DNA containing the cyclobutane pyrimidine dimer (CPD) lesion. Both RPA-A and RPA-AB were found to bind non- specifically to both strands of normal and CPD- containing DNA duplexes. There were no differences observed when binding to normal DNA duplex was examined in the presence of the minimal DNA-binding domain of XPA (XPA-MBD). However, there is a drastic difference for CPD-damaged DNA duplex as both RPA-A and RPA-AB bind specifically to the undamaged strand. The strand-specific binding of RPA and XPA to the damaged duplex DNA shows that RPA and XPA play crucial roles in damage verification and guiding cleavage of damaged DNA during NER.
在哺乳动物细胞中，核苷酸切除修复（NER）是去除大分子DNA加合物的主要途径。许多关键的NER蛋白是XP家族的成员（XPA、XPB等），该家族是根据其与色素性干皮病的关联而命名的。人类复制蛋白A（RPA）是普遍存在的单链DNA结合蛋白，是NER的另一种必需蛋白。RPA通过在受损DNA位点形成RPA-XPA复合物来刺激XPA与受损DNA的相互作用。在NER过程中，RPA与未受损DNA链的结合最为重要，因为指导切除核酸酶XPG和XPF的XPA必须与受损链结合。在本研究中，利用核磁共振（NMR）光谱来评估串联高亲和力DNA结合结构域RPA-AB以及分离结构域RPA-A与正常DNA和含有环丁烷嘧啶二聚体（CPD）损伤的受损DNA的结合情况。发现RPA-A和RPA-AB均非特异性地结合正常DNA双链体和含CPD的DNA双链体的两条链。在XPA的最小DNA结合结构域（XPA-MBD）存在的情况下检测与正常DNA双链体的结合时，未观察到差异。然而，对于CPD损伤的DNA双链体则存在显著差异，因为RPA-A和RPA-AB均特异性地结合未受损链。RPA和XPA与受损双链DNA的链特异性结合表明，RPA和XPA在NER过程中的损伤验证和指导受损DNA切割中发挥着关键作用。
