Abstract
DNA polymerase delta was purified from human placenta and its polymerase catalytic subunit identified as a 125-kDa polypeptide by activity staining. This 125-kDa form of DNA polymerase delta resembles that reported from calf thymus (Lee, M. Y. W. T., Tan, C.-K., Downey, K. M., and So, A. G. (1984) Biochemistry 23, 1906-1913) and differs in molecular properties from a previously described form isolated from human placenta (Lee, M. Y. W. T., and Toomey, N. L. (1987) Biochemistry 26, 1076-1085) and now referred to as DNA polymerase epsilon. The properties of DNA polymerase delta were further investigated to determine its relationships with DNA polymerase epsilon. The two enzymes differed in their response to proliferating cell nuclear antigen. Monoclonal antibodies against DNA polymerase delta were raised and used to examine its immunochemical relationships with DNA polymerase alpha and epsilon. These studies provided evidence that all three proteins are structurally distinct but share a common epitope(s). Immunofluorescence microscopy indicates that DNA polymerase delta and possibly also DNA polymerase epsilon are localized to the nucleus.
Highlights
KDa form of DNApolymerase6 resemblesthat reported DNA synthesis, since PCNA ias cell cycle associated protein from calf thymus
Other reports have indicated the presence of multiple formsof DNA polymerase 6 from calf thymus (Crute et al, 1986; Focher et al, 1988, 1989)
Inaddition,notall forms of DNA polymerase 6 display the sensitivityto PCNA reported for the 125-kDa foromf calf thymus DNApolymerase 6
Summary
DNA polymerase 6 activity was assayed usrng polyld.Al/ollgo~dTiln the absence. PH 7.0 and loaded onto a phosphocellulose column (1 6 x 3 cm) previously equllibrated with the same buffer. T h e column was eluted with a gradlent of KC1 10.1 M to 0.5 M. total volume 320 mll. The enzyme lracuons from thesecond peak were purlfled to homogenelty and was characterlzed as DNA polymerase a wlth a caralyuc subunit of 180 kDa IToomcy and Lee, unpubllshed observationsl. The pooled fractlons were chromatographed on an AN-gel Heparln column ( 3 x 7 cm). The pooled fractions from Step 7 were dlalyzed aganst 20 mM potasslum phosphate. The enzyme was eluted from the column with 0 . The dlalyzed e-s was loaded onto a Mono-Q Sf5 AEnryme activity was assayed in thperesence pol).ldAiloligoidTI I20.II a6 the trmplate of 100
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
