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J. Biol. Chem., Vol. 277, Issue 51, 50008-50014, December 20, 2002
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B*
,
From the Department of Pharmacological Sciences, State University
of New York at Stony Brook, Stony Brook, New York 11794-8651
We have recently reported the crystal structure
of the accessory subunit of mitochondrial DNA polymerase, pol 
B, 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 B 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 B dimer. A heterodimer of pol B 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 B dimer. The ability to bind
double-stranded DNA is not essential for pol B stimulation of pol
A activity in vitro, but may play a role in DNA
replication or repair.
Present address, Laboratory of Neurobiology, Dept. of Anatomy,
Embryology and Genetics, University of Zaragoza, Zaragoza
E-50013, Spain.
§
To whom correspondence should be addressed. Tel.: 631-444-3068;
Fax: 631-444-3218; E-mail: dan@pharm.sunysb.edu.
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