Melle Christian, Nasheuer Heinz-Peter
Institut für Molekulare Biotechnologie e.V., Abteilung Biochemie, Beutenbergstrasse 11, D-07745 Jena, Germany.
Nucleic Acids Res. 2002 Apr 1;30(7):1493-9. doi: 10.1093/nar/30.7.1493.
The wild-type form of p53 contains an intrinsic 3'-5'-exonuclease activity. As p53 forms a complex with DNA polymerase alpha-primase (pol-prim) in vivo this finding suggests that p53 might cooperate with pol-prim to stabilize the genetic information of living cells. To test this hypothesis, exonuclease-free DNA pol-prim was expressed alone or together with p53 for purification. Pol-prim formed a complex with p53, which was purified by ion exchange and immunoaffinity chromatography from baculovirus-infected insect cells. The p53-containing pol-prim fractions removed a 3'-unpaired nucleotide with a 1.5-2-fold higher rate than a paired nucleotide, whereas the four subunit pol-prim did not have any exonuclase activity. Therefore, only p53/pol-prim was able to elongate a primer-template that contained a 3'-unpaired primer end in vitro. To achieve this, the 3'-5'-exonuclease activity of p53 excised the unpaired nucleotide at the 3'-end of the primer and created a paired 3'-end, which pol-prim was able to elongate. The exonuclease activity of p53 as well as the elongation of a primer with a mispaired 3'-end was inhibited specifically by the anti-p53 monoclonal antibodies PAb240 and PAb421.
野生型p53具有内在的3'-5'-核酸外切酶活性。由于p53在体内与DNA聚合酶α-引发酶（pol-prim）形成复合物，这一发现表明p53可能与pol-prim协同作用以稳定活细胞的遗传信息。为了验证这一假设，单独或与p53一起表达无核酸外切酶的DNA pol-prim以进行纯化。Pol-prim与p53形成复合物，该复合物通过离子交换和免疫亲和色谱从杆状病毒感染的昆虫细胞中纯化出来。含p53的pol-prim组分去除3'-未配对核苷酸的速率比配对核苷酸高1.5至2倍，而四亚基pol-prim没有任何核酸外切酶活性。因此，只有p53/pol-prim能够在体外延伸含有3'-未配对引物末端的引物模板。为了实现这一点，p53的3'-5'-核酸外切酶活性切除引物3'-末端的未配对核苷酸并产生配对的3'-末端，pol-prim能够延伸该末端。p53的核酸外切酶活性以及具有错配3'-末端的引物的延伸被抗p53单克隆抗体PAb240和PAb421特异性抑制。
