Stimulation of human DNA polymerase epsilon by MDM2.
The human DNA polymerase epsilon catalytic subunit consists of a 140-kDa N-terminal domain that contains the catalytic activity and a 120-kDa C-terminal domain that binds to the other subunits and to exogenous peptides, including PCNA and MDM2. We report here that recombinant human MDM2 purified from insect cells or Escherichia coli stimulated the activity of DNA polymerase epsilon up to 10- and 40-fold, respectively, but not those of DNA polymerase beta or Klenow fragment of E.coli DNA polymerase I. Kinetic studies indicated that MDM2 increased the maximum velocity of the reaction, but did not change substrate affinities. The stimulation depended upon the interaction of the N-terminal 166 amino acid residues of MDM2 with the C-terminal domain of the full-length catalytic subunit, since the deletion of 166 amino acids from N-terminal of MDM2 or the removal of the C-terminal domain of DNA polymerase epsilon by trypsin digestion or competition for binding to it by the addition of excess C-terminal fragment eliminated the stimulation. Since DNA polymerase epsilon appears to be involved in DNA replication, recombination and repair synthesis, we suggest that MDM2 binding to DNA polymerase epsilon might be part of a reconfiguration process that allows DNA polymerase epsilon to associate with repair/recombination proteins in response to DNA damage.
Asahara
Hitomi
H
Division of Biochemistry and Molecular Biology, Barker Hall, University of California, Berkeley, CA 94720-3202, USA.
Li
Ying
Y
Fuss
Jill
J
Haines
Dale S
DS
Vlatkovic
Nikolina
N
Boyd
Mark T
MT
Linn
Stuart
S
D000818:Animals; D002460:Cell Line; D004257:DNA Polymerase II / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D009687:Nuclear Proteins; D010446:Peptide Fragments / Q000302:isolation & purification / Q000378:metabolism; D011485:Protein Binding; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051736:Proto-Oncogene Proteins c-mdm2; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D018411:Spodoptera; D013379:Substrate Specificity
D009687:Nuclear Proteins; D010446:Peptide Fragments; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2; D004257:DNA Polymerase II