Use of 2-aminopurine fluorescence to study the role of the beta hairpin in the proofreading pathway catalyzed by the phage T4 and RB69 DNA polymerases.
For DNA polymerases to proofread a misincorporated nucleotide, the terminal 3-4 nucleotides of the primer strand must be separated from the template strand before being bound in the exonuclease active center. Genetic and biochemical studies of the bacteriophage T4 DNA polymerase revealed that a prominent beta-hairpin structure in the exonuclease domain is needed to efficiently form the strand-separated exonuclease complexes. We present here further mutational analysis of the loop region of the T4 DNA polymerase beta-hairpin structure, which provides additional evidence that residues in the loop, namely, Y254 and G255, are important for DNA replication fidelity. The mechanism of strand separation was probed in in vitro reactions using the fluorescence of the base analogue 2-aminopurine (2AP) and mutant RB69 DNA polymerases that have modifications to the beta hairpin, to the exonuclease active site, or to both. We propose from these studies that the beta hairpin in the exonuclease domain of the T4 and RB69 DNA polymerases functions to facilitate strand separation, but residues in the exonuclease active center are required to capture the 3' end of the primer strand following strand separation.
Subuddhi
Usharani
U
Department of Biological Sciences, University of Alberta, Edmonton, Alberta T6G 2E9, Canada.
Hogg
Matthew
M
Reha-Krantz
Linda J
LJ
D015075:2-Aminopurine; D017122:Bacteriophage T4 / Q000201:enzymology* / Q000235:genetics*; D001665:Binding Sites; D002384:Catalysis; D019951:DNA Polymerase beta / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004261:DNA Replication; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D014764:Viral Proteins / Q000737:chemistry
D014764:Viral Proteins; D015075:2-Aminopurine; D019951:DNA Polymerase beta