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.

Polymerases:

Topics:

Historical Protein Properties (MW, pI, ...), Source / Purification, Nucleotide Incorporation

Status:

new topics/pols set partial results complete validated

Results:

Polymerase Reference Property Result Context
Human Pol lamba Shimazaki N2002 Molecular Weight 68kD Technique: Gel Filtration
Human Pol lamba Shimazaki N2002 Cloned or native Cloned in E. coli
Human Pol lamba Shimazaki N2002 Tagged Yes
Human Pol lamba Shimazaki N2002 Tag Name His
Human Pol lamba Shimazaki N2002 Full length or truncated Full length
Human Pol lamba Shimazaki N2002 Processivity 1bp
Human Pol lamba Shimazaki N2002 Specific Activity 3.37 units/mg Technique: Polymerase assay (poly[d(AT)]

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