The Asp285 variant of DNA polymerase beta extends mispaired primer termini via increased nucleotide binding.
Endogenous DNA damage occurs at a rate of at least 20,000 lesions per cell per day. Base excision repair (BER) is a key pathway for maintaining genome stability. Several pol beta variants were identified as conferring resistance to 3'-azido-3'-deoxythymidine (AZT) in Escherichia coli (Kosa et al. (1999) J. Biol. Chem. 274, 3851-3858). Detailed biochemical studies on one of these AZT-resistant variants, His285 to Asp, have shown that the H285D variant of pol beta possesses pre-steady-state kinetics that are similar to the wild-type polymerase. In gap filling assays with 5-bp gapped DNA, H285D showed a slight mutator phenotype. In depth single turnover kinetic analysis revealed that H285D is much more efficient than wild-type pol beta at extending mispaired primer termini. This mispair extension property of H285D is attributed to a greatly increased binding to the next correct nucleotide in the presence of a mispair. This change in K d(dNTP),app is not accompanied by a change in k pol; values for k pol are the same for both H285D and wild-type. Close examination of available structural data, as well as molecular modeling, has shown that residue 285 is able to make several stabilizing contacts in the fingers domain of the polymerase, and the introduction of a negatively charged side chain could have important effects on the enzyme. It is postulated that the loss of the contact between His285, Lys289, and Ile323 is responsible for the ability of H285D to extend mispairs through disruption of contacts near the C-terminal end of pol beta and propagation into the nucleotide binding pocket.
Murphy
Drew L
DL
Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, Connecticut 06520, USA.
Kosa
Jessica
J
Jaeger
Joachim
J
Sweasy
Joann B
JB
D019943:Amino Acid Substitution; D001224:Aspartic Acid / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002942:Circular Dichroism; D003198:Computer Simulation; D019951:DNA Polymerase beta / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009711:Nucleotides / Q000378:metabolism*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D013379:Substrate Specificity
D009711:Nucleotides; D001224:Aspartic Acid; D019951:DNA Polymerase beta