Cloning, expression and characterization of human tissue-specific DNA polymerase lambda2.

Abstract:

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 lambda2 (GenBank Accession No. AY302442). POL lambda2 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 lambda2 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 lambda2 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 lambda2 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 lambda2 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 chromatography in an FPLC system. The analysis using isotope a-(32)P-dCTP incorporation in vitro showed that the purified recombinant POL lambda2 exhibited DNA polymerase activity.

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