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

DNA polymerase (POL) lambda plays an important role during DNA repair and DNA nonhomologous recombination processes. A novel POL lambda variant was cloned from a human liver cDNA library and named POL lambda 2 (GenBank Accession No. AY302442). POL lambda 2 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 lambda 2 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 lambda 2 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 lambda and POL lambda 2 in normal liver tissues and hepatocellular carcinoma (HCC) adjacent tissues, the ratio was aberrant in 80% of those 15 HCC specimens examined due to the up-regulated expression of POL lambda. This abnormality might be involved in hepatocarcinogenesis. The recombinant POL \2 with His-tag was expressed as a soluble active protein in E. coli BL21 (M)CONDON Plus and purified by Ni-NTA resin and then desalted by Superdex-75 chromatography in an FPLC system. The analysis using isotope a-P-32-dCTP incorporation in vitro showed that the purified recombinant POL lambda 2 exhibited DNA polymerase activity.