Jónsson Z O, Hindges R, Hübscher U
Department of Veterinary Biochemistry, University Zürich-Irchel, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland.
EMBO J. 1998 Apr 15;17(8):2412-25. doi: 10.1093/emboj/17.8.2412.
The DNA polymerase accessory factor proliferating cell nuclear antigen (PCNA) has been caught in interaction with an ever increasing number of proteins. To characterize the sites and functions of some of these interactions, we constructed four mutants of human PCNA and analysed them in a variety of assays. By targeting loops on the surface of the PCNA trimer and changing three or four residues at a time to alanine, we found that a region including part of the domain-connecting loop of PCNA and loops on one face of the trimer, close to the C-termini, is involved in binding to all of the following proteins: DNA polymerase delta, replication factor C, the flap endonuclease Fen1, the cyclin dependent kinase inhibitor p21 and DNA ligase I. An inhibition of DNA ligation caused by the interaction of PCNA with DNA ligase I was found, and we show that DNA ligase I and Fen1 can inhibit DNA synthesis by DNA polymerase delta/PCNA. We demonstrate that PCNA must be located below a 5' flap on a forked template to stimulate Fen1 activity, and considering the interacting region on PCNA for Fen1, this suggests an orientation for PCNA during DNA replication with the C-termini facing forwards, in the direction of DNA synthesis.
DNA聚合酶辅助因子增殖细胞核抗原（PCNA）已被发现与越来越多的蛋白质相互作用。为了表征其中一些相互作用的位点和功能，我们构建了四种人类PCNA突变体，并在各种分析中对其进行了分析。通过靶向PCNA三聚体表面的环，并一次将三个或四个残基突变为丙氨酸，我们发现一个区域，包括PCNA结构域连接环的一部分以及三聚体一侧靠近C末端的环，参与与以下所有蛋白质的结合：DNA聚合酶δ、复制因子C、瓣状核酸内切酶Fen1、细胞周期蛋白依赖性激酶抑制剂p21和DNA连接酶I。我们发现PCNA与DNA连接酶I的相互作用会导致DNA连接受到抑制，并且我们表明DNA连接酶I和Fen1可以抑制DNA聚合酶δ/PCNA的DNA合成。我们证明PCNA必须位于叉状模板上5'瓣的下方才能刺激Fen1活性，考虑到PCNA与Fen1的相互作用区域，这表明在DNA复制过程中PCNA的C末端朝前，朝着DNA合成的方向。
