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

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Abstract

DNA polymerase (POL) λ plays an important role during DNA repair and DNA nonhomologous recombination processes. A novel 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 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 λ. This abnormality might be involved in hepatocarcinogenesis. 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 chromatography in an FPLC system. The analysis using isotope a-32P-dCTP incorporation in vitro showed that the purified recombinant POL λ2 exhibited DNA polymerase activity.

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References

  1. Hübscher U, Maga G, Spadari S. Eukaryotic DNA polymerases. Annu Rev Biochem, 2002, 71: 133–163

    Article  PubMed  Google Scholar 

  2. Aoufouchi S, Flatter E, Dahan A, et al. Two novel human and mouse DNA polymerases of the polX family. Nucleic Acids Res, 2000, 28: 3684–3693

    Article  PubMed  CAS  Google Scholar 

  3. Garcia-Diaz M, Dominguez O, Lopez-Fernandez L A, et al. DNA polymerase lambda (Pol lambda), a novel eukaryotic DNA polymerase with a potential role in meiosis. J Mol Biol, 2000, 301: 851–867

    Article  PubMed  CAS  Google Scholar 

  4. Nagasawa K, Kitamura K, Yasui A, et al. Identification and characterization of human DNA polymerase beta 2, A DNA polymerase beta-related enzyme. J Biol Chem, 2000, 275: 31233–31238

    Article  PubMed  CAS  Google Scholar 

  5. Bork P, Hofmann K, Bucher P, et al. A superfamily of conserved domains in DNA damage-responsive cell cycle checkpoint proteins. FASEB J, 1997, 11: 68–76

    PubMed  CAS  Google Scholar 

  6. Doherty A J, Serpell L C, Ponting C P. The helix-hairpin-helix DNA-binding motif: A structural basis for non-sequence-specific recognition of DNA. Nucleic Acids Res, 1996, 24: 2488–2497

    Article  PubMed  CAS  Google Scholar 

  7. Ramadan K, Shevelev I, Hübsche U. The DNA-polymerase-X family: Controllers of DNA quality? Nat Rev Mol Cell Biol, 2004, 5: 1038–1043

    Article  PubMed  CAS  Google Scholar 

  8. Lee J W, Blanco L, Zhou T, et al. Implication of DNA polymerase lambda in alignment-based gap filling for nonhomologous DNA end joining in human nuclear extracts. J Biol Chem, 2004, 279: 805–811

    Article  PubMed  CAS  Google Scholar 

  9. Ramadan K, Maga G, Shevelev I V, et al. Human DNA polymerase lambda possesses terminal deoxyribonucleotidyl transferase activity and can elongate RNA primers: Implications for novel functions. J Mol Biol, 2003, 328: 63–72

    Article  PubMed  CAS  Google Scholar 

  10. Kedar P S, Kim S J, Robertson A, et al. Direct interaction between mammalian DNA polymerase beta and proliferating cell nuclear antigen. J Biol Chem, 2002, 277: 31115–31123

    Article  PubMed  CAS  Google Scholar 

  11. Bebenek K, Garcia-Diaz M, Blanco L, et al. The frameshift infidelity of human DNA polymerase lambda. Implications for function. J Biol Chem, 2003, 278: 34685–34690

    Article  PubMed  CAS  Google Scholar 

  12. Maga G, Sheveley I, Villani G, et al. Human replication protein A can suppress the intrinsic in vitro mutator phenotype of human DNA polymerase lambda. Nucleic Acids Res, 2006, 34: 1405–1415

    Article  PubMed  CAS  Google Scholar 

  13. Shimazaki N, Yoshida K, Kobayashi T, et al. Over-expression of human DNA polymerase lambda in E. coli and characterization of the recombinant enzyme. Genes Cells, 2002, 7: 639–651

    Article  PubMed  CAS  Google Scholar 

  14. Mizushina Y, Kamisuki S, Kasai N, et al. Petasiphenol: A DNA polymerase lambda inhibitor. Biochemistry, 2002, 41: 14463–14471

    Article  PubMed  CAS  Google Scholar 

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Author notes

  1. Gu Fu

    Present address: Food Inspection and Supervision Institute of Shanxi Province, No. 222 Changzhi Road, Taiyuan, 030012, China

Authors and Affiliations

  1. State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai, 200433, China

    Gu Fu, You Chun, Liu JianPing, Chen Ao, Yu Yao, Wang Xiang, Yuan HanYing, Li YuYang & Lü Hong

  2. State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Shanghai, 200032, China

    Wan DaFang & Gu JianRen

Authors
  1. Gu Fu
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  2. You Chun
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  3. Liu JianPing
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  4. Chen Ao
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  5. Yu Yao
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  6. Wang Xiang
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  7. Wan DaFang
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  8. Gu JianRen
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  9. Yuan HanYing
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  10. Li YuYang
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  11. Lü Hong
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Corresponding author

Correspondence to Lü Hong.

Additional information

Supported by the National Natural Science Foundation of China (Grant No. 30370752), Shanghai Key Technologies R&D Program of Biomedicine (Grant No. 05dz19302) and CNHLPP (Grant No. 2004BA711A19)

Recommended by Prof. SHEN Yan, member of the editorial board of Science in China

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Gu, F., You, C., Liu, J. et al. Cloning, expression and characterization of human tissue-specific DNA polymerase λ2. SCI CHINA SER C 50, 457–465 (2007). https://doi.org/10.1007/s11427-007-0059-4

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  • DOI: https://doi.org/10.1007/s11427-007-0059-4

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