title: Over-expression of human DNA polymerase lambda in E. coli and characterization of the recombinant enzyme.
abstract: BACKGROUND
  DNA polymerase lambda (Pol lambda) was recently identified as a new member of the family X of DNA polymerases in eukaryotic cells. Pol lambda contains a nuclear localization signal (NLS), a BRCA1-C terminal (BRCT) domain, a proline-rich region, helix-hairpin-helix (HhH) and pol X motifs. Since the amino acid sequence for Pol lambda shares a high degree of homology to Pol beta, Pol lambda is considered to have a similar enzymatic nature to Pol beta.
  
  
  RESULTS
  Recombinant human Pol lambda was shown to possess template-directed DNA polymerase activity in its monomeric form. Pol lambda required either Mn2+ or Mg2+ as a metal co-factor to catalyse this activity, and optimal activity was detected at pH 8.5-9.0. Pol lambda was insensitive to aphidicolin, but was sensitive to dideoxynucleoside triphosphates or N-ethylmaleimide. By constructing the truncated Pol lambda, the proline rich region was shown to act in a suppression of its polymerization activity. A chimeric enzyme comprised of the Pol lambda N-terminal region and Pol beta also showed a reduced Pol beta activity. Proliferating cell nuclear antigen (PCNA) directly interacts with Pol lambda through its Pol beta like region in vitro.
  
  
  CONCLUSIONS
  Pol lambda possesses similar enzymatic nature to Pol beta; requirements of cations and optimal conditions for pH and NaCl concentration, aside from sensitivity to N-ethylmaleimide and template preference. The proline rich region of Pol lambda functions as a suppressor domain for its polymerization activity (SDPA). Pol lambda interacts directly with PCNA through its Pol beta like region. The functional consequence of this interaction is the negative regulation of Pol lambda activity.
authors.affiliation: Department of Applied Biological Science, Faculty of Science and Technology, Science University of Tokyo, Noda, Chiba 278-8510, Japan.
authors.forename: Noriko
authors.initials: N
authors.lastname: Shimazaki
authors.forename: Kenta
authors.initials: K
authors.lastname: Yoshida
authors.forename: Toshiko
authors.initials: T
authors.lastname: Kobayashi
authors.forename: Shingo
authors.initials: S
authors.lastname: Toji
authors.forename: Katsuyuki
authors.initials: K
authors.lastname: Tamai
authors.forename: Osamu
authors.initials: O
authors.lastname: Koiwai
mesh_terms: D001665:Binding Sites; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism; D019951:DNA Polymerase beta / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli; D006801:Humans; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D018809:Proliferating Cell Nuclear Antigen; D011993:Recombinant Fusion Proteins; D004247:DNA; C415367:DNA polymerase beta2; D019951:DNA Polymerase beta