DNA binding properties of human pol gammaB.
We have recently reported the crystal structure of the accessory subunit of mitochondrial DNA polymerase, pol gammaB, and identified a region of the protein involved in DNA binding. The DNA employed in previous studies was presumed to be single-stranded, because it was generated by single-sided PCR. Further characterization of this DNA indicated that, due to a strand transfer event during synthesis by single-sided PCR, the DNA adopts a double-stranded hairpin conformation under native conditions. We used a series of double- and single-stranded oligonucleotides of different lengths to confirm that human pol gammaB prefers to bind double-stranded DNA longer than 40 bp with little apparent sequence specificity. Site-specific deletion mutagenesis identified clusters of basic residues in two surface loops required for DNA binding located on opposite sides of the symmetrical pol gammaB dimer. A heterodimer of pol gammaB that contains one mutant and one wild-type DNA binding region was shown to be unable to bind double-stranded DNA, suggesting that a single DNA molecule must contact both DNA binding sites in the pol gammaB dimer. The ability to bind double-stranded DNA is not essential for pol gammaB stimulation of pol gammaA activity in vitro, but may play a role in DNA replication or repair.
Carrodeguas
José A
JA
Department of Pharmacological Sciences, State University of New York at Stony Brook, Stony Brook, NY 11794-8651, USA.
Pinz
Kevin G
KG
Bogenhagen
Daniel F
DF
D001483:Base Sequence; D020134:Catalytic Domain; D004247:DNA / Q000378:metabolism; D000074002:DNA Polymerase gamma; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000378:metabolism*; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D017353:Gene Deletion; D006801:Humans; D008957:Models, Genetic; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D009841:Oligonucleotides / Q000737:chemistry; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011687:Purines / Q000737:chemistry; D011994:Recombinant Proteins / Q000378:metabolism
D009841:Oligonucleotides; D011687:Purines; D011994:Recombinant Proteins; D004247:DNA; D000074002:DNA Polymerase gamma; D004259:DNA-Directed DNA Polymerase