Isolation and identification of the third subunit of mammalian DNA polymerase delta by PCNA-affinity chromatography of mouse FM3A cell extracts.
Using proliferating cell nuclear antigen affinity chroma-tography and glycerol gradient centrifugation of partially purified fractions from mouse FM3A cells we have been able to isolate novel complexes of DNA polymerase delta and DNA ligase 1 containing clearly defined subunit compositions. In addition to the well known catalytic subunit of 125 kDa and accessory subunit of 48 kDa, the DNA polymerase delta complex contained three supplementary components, one of which reacted with antibodies directed against the p40 and p37 subunits of RF-C. Of the two remaining components, one termed p66 turned out to be coded by a gene whose putative C-terminal domain displayed significant homology with that of the Cdc27 subunit of Schizosaccharomyces pombe polymerase delta. On the basis of these and other observations, we propose p66 to be the missing third subunit of mammalian DNA polymerase delta. The DNA ligase 1 complex was made up of three novel components in addition to the 125 kDa catalytic subunit, two of which, p48 and p66, were common to DNA polymerase delta. We discuss the implications of our findings within the current framework of our understanding of DNA replication.
Hughes
P
P
Centre National de la Recherche Scientifique (CNRS), UPR9044, Institut de Recherches sur le Cancer,7 rue Guy Moquet BP 8, 94801 Villejuif, France. hughes@infobiogen.fr
Tratner
I
I
Ducoux
M
M
Piard
K
K
Baldacci
G
G
D000595:Amino Acid Sequence; D000818:Animals; D064191:Apc3 Subunit, Anaphase-Promoting Complex-Cyclosome; D001483:Base Sequence; D018797:Cell Cycle Proteins / Q000235:genetics; D002460:Cell Line; D002846:Chromatography, Affinity / Q000379:methods*; D004258:DNA Polymerase III / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D004274:DNA, Recombinant / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D018809:Proliferating Cell Nuclear Antigen / Q000235:genetics; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000235:genetics; D012568:Schizosaccharomyces / Q000235:genetics; D029702:Schizosaccharomyces pombe Proteins; D017386:Sequence Homology, Amino Acid
D064191:Apc3 Subunit, Anaphase-Promoting Complex-Cyclosome; C577366:CDC27 protein, human; D018797:Cell Cycle Proteins; D004274:DNA, Recombinant; D005656:Fungal Proteins; D018809:Proliferating Cell Nuclear Antigen; D011994:Recombinant Proteins; D029702:Schizosaccharomyces pombe Proteins; C513024:cdc27 protein, S pombe; C435720:POLD3 protein, human; D004258:DNA Polymerase III