Loop II of DNA polymerase beta is important for discrimination during substrate binding.
Loop II of DNA polymerase beta (pol beta) consists of 14 amino acid residues and is highly flexible and solvent exposed. Previous research from our laboratory has shown that this loop is important for polymerase activity and fidelity. In the study presented here, we demonstrate that a shortened five amino acid residue loop compromises the fidelity of pol beta. This five-residue loop, termed ENEYP, induces one base frameshift errors and A-C transversions within a specific sequence context. We demonstrate that ENEYP misincorporates dGTP opposite template A at higher efficiencies than wild-type pol beta. The kinetic basis for misincorporation is a defect in discrimination of the correct from incorrect dNTP substrate at the level of ground-state binding. Our results are consistent with the idea that loop II of pol beta functions to maintain accurate DNA synthesis by a direct or indirect influence on the nucleotide binding pocket.
Lin
George C
GC
Department of Therapeutic Radiology and Genetics, Yale University School of Medicine, 333 Cedar St., New Haven, CT 06520, USA.
Jaeger
Joachim
J
Eckert
Kristin A
KA
Sweasy
Joann B
JB
D000241:Adenosine / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D020137:Base Pair Mismatch; D001483:Base Sequence; D004247:DNA / Q000378:metabolism; D019951:DNA Polymerase beta / Q000737:chemistry* / Q000378:metabolism*; D016368:Frameshift Mutation; D006160:Guanosine Triphosphate / Q000378:metabolism; D018259:Herpesvirus 1, Human / Q000201:enzymology; D007700:Kinetics; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D017433:Protein Structure, Secondary; D051381:Rats; D017384:Sequence Deletion; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013937:Thymidine Kinase / Q000378:metabolism
D050505:Mutant Proteins; D006160:Guanosine Triphosphate; D004247:DNA; D013937:Thymidine Kinase; D019951:DNA Polymerase beta; D000241:Adenosine