Effects of Xenopus laevis Mitochondrial Single-stranded DNA-binding Protein on Primer-Template Binding and 3′→5′ Exonuclease Activity of DNA Polymerase γ

Abstract

Mitochondrial DNA (mtDNA) is replicated by DNA polymerase ␥ by a strand displacement mechanism involving mitochondrial single-stranded DNA-binding protein (mtSSB). mtSSB stimulates the overall rate of DNA synthesis on singly-primed M13 DNA mainly by stimulating the processivity of DNA synthesis rather than by stimulating primer recognition. We used electrophoretic mobility shift methods to study the effects of mtSSB on primer-template recognition by DNA pol ␥. Preliminary experiments showed that single mtSSB tetramers bind tightly to oligo(dT) single strands containing 32 to 48 residues. An oligonucleotide primer-template was designed with an 18-mer primer annealed to the 3-portion of a 71-mer template containing 40 dT residues at its 5-end as a binding site for mtSSB. DNA pol ␥ bound to this primer-template either in the absence or presence of mtSSB in complexes that remained intact and enzymatically active following native gel electrophoresis. Association of mtSSB with the 5-dT 40 -tail in the 18:71-mer primer-template reduced the binding of DNA polymerase ␥ and the efficiency of primer extension. Binding of mtSSB to single-stranded DNA was also observed to block the action of the 335 exonuclease of DNA polymerase ␥. The size of fragments protected from 335 exonuclease trimming increases with increasing ionic strength in a manner consistent with the known salt dependence of the binding site size of Escherichia coli SSB.Single-stranded DNA-binding proteins (SSB) 1 comprise a group of proteins which bind preferentially and with high affinity to single-stranded DNA. SSB proteins are present in prokaryotic and eukaryotic cells and are assumed to be essential for DNA metabolism in all organisms. One of the best characterized prokaryotic SSBs, Escherichia coli SSB, is a stable homotetramer of subunits containing 177 amino acids (18.8 kDa). Both biochemical and genetic data indicate that E. coli SSB is involved in DNA replication, repair, and recombination (1). Depending on the ionic conditions this protein binds to DNA in multiple modes with an apparent binding site size varying from 35 to 56 or 65 nucleotides (2, 3). Eukaryotes contain two different families of nuclear and mitochondrial SSB proteins. Heterotrimeric nuclear SSBs (RPA) (subunits of about 70, 32, and 14 kDa) have been found in all eukaryotic cells examined. These proteins are essential for replication, are involved in recombination and repair, and interact specifically with other proteins involved in DNA metabolism although they have little sequence homology with prokaryotic SSBs (4).Mitochondrial SSBs have been isolated from Xenopus laevis oocytes (5), rat liver (6, 7), yeast (8), and Drosophila (9). Two Xenopus mtSSBs have been described. One of these, mtSSB-1 has been fully sequenced (10) and is represented in a fulllength cDNA clone (11). The second, mtSSB-2, has not been cloned and has been only partially sequenced. The first 80 residues of mtSSB-2 reveal 91% identity to mtSSB-1, which contains 129 amino acids (14.6 kDa). Both Xenopus (12) and ...

The Accessory Subunit of Xenopus laevis Mitochondrial DNA Polymerase γ Increases Processivity of the Catalytic Subunit of Human DNA Polymerase γ and Is Related to Class II Aminoacyl-tRNA Synthetases

“…We have previously shown that authentic Xenopus DNA pol ␥ binds to oligonucleotide substrates and retains polymerase activity following native gel electrophoresis (23). Binding of the human catalytic subunit alone produced a shifted complex with a more rapid gel mobility than that observed for the Xenopus pol ␥ holoenzyme (compare lanes 2 and 3 in Fig.…”

“…The homopolymeric template was included in this analysis since we have previously shown that this is a preferred template for DNA pol ␥ because it does not present the enzyme with blocks to replication that require a mitochondrial single-stranded DNA binding protein (SSB) as an additional processivity factor (23). In each case, the primers were 32 P end labeled for the primer extension assays.…”

“…DNA pol ␥ was purified from X. laevis ovary mitochondria as previously described (15,23). SDS-PAGE analysis of the purified enzyme revealed the presence of two polypeptides, of approximately 140 and 45 kDa (Fig.…”

“…Purification of X. laevis DNA pol ␥ has been described elsewhere (15,23). Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) separated two polypeptides of 140 and 45 kDa.…”

Characterization of a Catalytically Slow AP Lyase Activity in DNA Polymerase γ and Other Family A DNA Polymerases

“…The preparation from Roche Molecular Biochemicals was selected for repair assays. DNA pol ␥ was purified from an Xenopus laevis ovary homogenate as described (19,20). The preparation used in this study had a specific activity of 30 units/ml where 1 unit corresponds to incorporation of 1 nmol of TMP on a poly(dA)-oligo(dT) template-primer in 60 min at 30°C.…”