Isolation and identification of the third subunit of mammalian DNA polymerase δ by PCNA-affinity chromatography of mouse FM3A cell extracts

• Published in: Nucleic acids research 1999-05, Vol.27 (10), p.2108-2114
• Main Authors: Hughes, Patrick, Tratner, Isabelle, Ducoux, Manuelle, Piard, Karine, Baldacci, Giuseppe
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Apc3 Subunit, Anaphase-Promoting Complex-Cyclosome; Base Sequence; Cell Cycle Proteins - genetics; Cell Line; Chromatography, Affinity - methods; DNA Polymerase III - chemistry; DNA Polymerase III - genetics; DNA Polymerase III - isolation & purification; DNA, Recombinant - genetics; Fungal Proteins - genetics; HeLa Cells; Humans; Mice; Molecular Sequence Data; Proliferating Cell Nuclear Antigen - genetics; Protein Conformation; Recombinant Proteins - genetics; Schizosaccharomyces - genetics; Schizosaccharomyces pombe Proteins; Sequence Homology, Amino Acid
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/27.10.2108

Using proliferating cell nuclear antigen affinity chromatography and glycerol gradient centrifugation of partially purified fractions from mouse FM3A cells we have been able to isolate novel complexes of DNA polymerase δ 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 δ 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 δ. On the basis of these and other observations, we propose p66 to be the missing third subunit of mammalian DNA polymerase δ. 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 δ. We discuss the implications of our findings within the current framework of our understanding of DNA replication.