In vitro DNA repair genomics using XR-seq with Escherichia coli and mammalian cell-free extracts.
In vitro DNA repair genomics using XR-seq with Escherichia coli and mammalian cell-free extracts.
复制标题
DOI：
10.1073/pnas.2314233120
复制
发表时间：
2023-10-24
影响因子：
11.1
通讯作者：
Sancar, Aziz
中科院分区：
文献类型：
--
作者：
Cao, Xuemei;Kose, Cansu;Selby, Christopher P.;Sancar, Aziz
由产生大体积加合物的UV光和化学试剂引起的DNA损伤通过核苷酸切除修复以长度为12-13（原核生物）和26-27（真核生物）碱基的含有损伤的寡核苷酸的形式被去除。XR-seq（eXcision Repair-sequencing）是一种开发用于从细胞或组织中捕获切除的寡核苷酸并在单核苷酸水平上将切除产物与基因组进行测序和比对的方法。在这里，我们描述了在大肠杆菌和哺乳动物细胞中进行XR-seq的体外方法，使用无细胞提取物和紫外线损伤的质粒。体外与体内XR-seq修复图谱的比较有望为DNA高阶结构和组蛋白修饰以及染色质化对DNA损伤形成和修复的影响提供新的见解。XR-seq（eXcision Repair-sequencing）方法已被广泛用于在从大肠杆菌到酵母、果蝇、拟南芥、小鼠和人类的生物体中绘制核苷酸切除修复全基因组图谱。该方法的基本特征是捕获切除的携带DNA损伤的寡聚体，对其进行测序，并将其序列与基因组进行比对。我们希望使用补充有受损DNA底物的无细胞提取物在体外进行XR-seq，以便在研究细胞背景下影响核苷酸切除修复的因素时具有更大的灵活性[M. J. Smerdon，J. J. Wyrick，S. Delaney，J.Biol.Chem.299，105118（2023）]。我们在这里报告了成功地使用紫外线照射质粒作为底物的修复在体外和体内的E。coli和E.大肠杆菌无细胞提取物和哺乳动物无细胞提取物。XR-seq分析证明了细菌和哺乳动物系统在体外和体内的共同切除产物长度和序列特征。这种方法有望帮助理解表观遗传学和其他细胞因素和条件对DNA修复的影响。
DNA damage caused by UV light and chemical agents that produce bulky adducts is removed in the form of damage-containing oligonucleotides 12–13 (prokaryotes) and 26–27 (eukaryotes) bases in length by nucleotide excision repair. XR-seq (eXcision Repair-sequencing) is a method developed to capture the excised oligonucleotides from cells or tissues and sequence and align the excision products to the genome at the single-nucleotide level. Here, we describe in vitro methods for XR-seq in both Escherichia coli and mammalian cells, using cell-free extracts and ultraviolet light-damaged plasmids. Comparison of in vitro with in vivo XR-seq repair maps are expected to provide novel insights on the effects of DNA higher order structure and histone modifications and chromatinization on DNA damage formation and repair. The XR-seq (eXcision Repair-sequencing) method has been extensively used to map nucleotide excision repair genome-wide in organisms ranging from Escherichia coli to yeast, Drosophila, Arabidopsis, mice, and humans. The basic feature of the method is to capture the excised oligomers carrying DNA damage, sequence them, and align their sequences to the genome. We wished to perform XR-seq in vitro with cell-free extract supplemented with a damaged DNA substrate so as to have greater flexibility in investigating factors that affect nucleotide excision repair in the cellular context [M. J. Smerdon, J. J. Wyrick, S. Delaney, J. Biol. Chem. 299, 105118 (2023)]. We report here the successful use of ultraviolet light-irradiated plasmids as substrates for repair in vitro and in vivo by E. coli and E. coli cell-free extracts and by mammalian cell-free extract. XR-seq analyses demonstrated common excision product length and sequence characteristics in vitro and in vivo for both the bacterial and mammalian systems. This approach is expected to help understand the effects of epigenetics and other cellular factors and conditions on DNA repair.
登录
查看更多内容
影响因子：
48
作者：
Langmead, Ben;Salzberg, Steven L.
通讯作者：
Salzberg, Steven L.
影响因子：
5.8
作者：
Barnett, Derek W.;Garrison, Erik K.;Marth, Gabor T.
通讯作者：
Marth, Gabor T.
DOI：
10.1073/pnas.1614430113
发表时间：
2016-10-11
影响因子：
11.1
作者：
Hu, Jinchuan;Lieb, Jason D.;Adar, Sheera
通讯作者：
Adar, Sheera
影响因子：
4.8
作者：
Adebali, Ogun;Sancar, Aziz;Selby, Christopher P.
通讯作者：
Selby, Christopher P.
影响因子：
14.9
作者：
Mu, Hong;Kropachev, Konstantin;Broyde, Suse
通讯作者：
Broyde, Suse