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Cloning, expression and characterization of human tissue-specific DNA polymerase λ2

Science china(Life Sciences) 2007年第4期50卷 457-465页

作者： GU Fu1, YOU Chun1, LIU JianPing1, CHEN Ao1,,YU Yao1, WANG Xiang1, WAN DaFang2, GU JianRen2, YUAN HanYing1, LI YuYang1 & Lü Hong1 1 state key laboratory of Genetic Engineering, institute of Genetics, School of Life Sciences, Fudan University, shanghai 200433, china 2 state key laboratory of oncogenes and related genes, shanghai cancer institute, shanghai 200032, china present address: food inspection and supervision institute of shanxi province, no. 222 changzhi road, taiyuan 030012, china 1. state key laboratory of Genetic Engineering Institute of Genetics School of Life Sciences Fudan University Shanghai 200433 China 2. state key laboratory of oncogenes and related genes Shanghai Cancer Institute Shanghai 200032 China

DNA polymerase (POL) λ plays an important role during DNA repair and DNA no.homologous recom-bination processes. A no.el POL λ variant was cloned from a human liver cDNA library and named POL λ2 (GenBank Accession no. AY302442). POL λ2 has 2206 base pairs in length with an open reading frame of 1452 base pairs encoding a 482-amino.acids protein. Bioinformatics analysis reveals that POL λ2 spans 7.9 kb on human chromosome 10q24 and is composed of 8 exons and 7 introns. It has the specific domain of DNA polymerase X family-POL Xc at the C-terminus and BRCT domain at the N-terminus. POL λ2 was localized predominantly in nucleus in transfected L0-2 cells. It was expressed abundantly in liver and testis, weakly in ovary, and undetectably in other tested human tissues. In comparison with the expression ratio between POL λ and POL λ2 in no.mal liver tissues and hepato-cellular carcino.a (HCC) adjacent tissues, the ratio was aberrant in 80% of those 15 HCC specimens examined due to the up-regulated expression of POL λ. This abno.mality might be involved in hepato-carcino.0}is. The recombinant POL λ2 with His-tag was expressed as a soluble active protein in E. coli BL21 (DE3)CONDON Plus and purified by Ni-NTA resin and then desalted by Superdex-75 chro-matography in an FPLC system. The analysis using isotope α-32P-dCTP incorporation in vitro showed that the purified recombinant POL λ2 exhibited DNA polymerase activity.

关键词： POL λ2 gene cloning expression purification DNA polymerase activity hepatocellular carcino.a

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