Parsons Jason L, Dianova Irina I, Dianov Grigory L
MRC Radiation and Genome Stability Unit, Harwell, Oxfordshire OX11 0RD, UK.
Nucleic Acids Res. 2004 Jul 6;32(12):3531-6. doi: 10.1093/nar/gkh676. Print 2004.
DNA strand breaks containing 3'-phosphoglycolate (3'-PG) ends are the major lesions induced by ionizing radiation. The repair of this lesion is not completely understood and several activities are thought to be involved in processing of 3'-PG ends. In this study we examined activities in human whole cell extracts (WCE) responsible for removal of 3'-PG. Using a radiolabelled oligonucleotide containing a single nucleotide gap with internal 5'-phosphate and 3'-PG ends, we demonstrate that the major 3'-PG activity in human WCE is Mg2+ dependent and that this activity co-purifies with AP endonuclease 1 (APE1) over phosphocellulose and gel filtration chromatography. Furthermore, immunodepletion of APE1 from active gel filtration fractions using APE1 specific antibodies reveals that the major activity against 3'-PG in human WCE is APE1.
含有3'-磷酸乙醇酸(3'-PG)末端的DNA链断裂是电离辐射诱导产生的主要损伤。这种损伤的修复机制尚未完全明确,人们认为有几种活性参与了3'-PG末端的处理过程。在本研究中,我们检测了人全细胞提取物(WCE)中负责去除3'-PG的活性。使用一种含有单个核苷酸缺口、内部带有5'-磷酸和3'-PG末端的放射性标记寡核苷酸,我们证明人WCE中的主要3'-PG活性依赖于Mg2+,并且该活性在磷酸纤维素柱和凝胶过滤层析中与脱嘌呤嘧啶内切酶1(APE1)共纯化。此外,使用APE1特异性抗体从活性凝胶过滤级分中免疫去除APE1后发现,人WCE中针对3'-PG的主要活性就是APE1。