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Nucleic Acids Res
2000 Mar 01;285:1237-44. doi: 10.1093/nar/28.5.1237.
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Protein sequences conserved in prokaryotic aminoacyl-tRNA synthetases are important for the activity of the processivity factor of human mitochondrial DNA polymerase.
Carrodeguas JA
,
Bogenhagen DF
.
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Previous studies have shown that the small subunit of Xenopus DNA polymerase gamma (pol gammaB) acts as a processivity factor to stimulate the 140 kDa catalytic subunit of human DNA polymerase gamma. A putative human pol gammaB initially identified by analysis of DNA sequence had not been shown to be functional, and appeared to be an incomplete clone. In this paper, we report the cloning of full-length human and mouse pol gammaB. Both human and mouse pol gammaB proteins were expressed in their mature forms, without their apparent mitochondrial localization signals, and shown to stimulate processivity of the recombinant catalytic subunit of human pol gammaA. Deletion analysis of human pol gammaB indicated that blocks of sequence conserved with prokaryotic class II aminoacyl-tRNA synthetases are necessary for activity and inter-action with human pol gammaA. Purification of DNA pol gamma from HeLa cells indicated that both proteins are associated in vivo.
Figure 1. Alignment of vertebrate pol γB subunits with T.Thermophilus GRS. The program Clustal (PCGene) was used to align the sequences of human (HSGB; GenBank accession number AF177201) , mouse (MMGB; accession number AF177202) and Xenopus (XLGB; accession number AF124606) pol γB subunits with T.thermophilus GRS (TTGRS; accession number P56206) . Identical amino acids are indicated with asterisks; similar ones, with dots. The first amino acid of the mature Xenopus protein and the predicted ones for the human and mouse proteins are indicated in bold. The consensus motifs in class II aaRS sequences are underlined in the TTGRS sequence (31). The DNA sequence we determined for human pol γB includes a few changes with respect to the original sequence reported in GenBank U94703, as follows: silent T to C changes at positions 371 and 998; C to G at 448, resulting in a T122 to R change in the protein sequence; A to G at 488, resulting in S136 changed to G; G to A in 587, resulting in A169 changed to T; and a short frame shift caused by an insertion of an A in position 940 and a deletion of a C in position 961, resulting in a change of sequence TNFTTI to NKLYYN.
Figure 2. All three vertebrate pol γB subunits stimulate processive DNA synthesis by human pol γA. (A) Coomassie blue stained SDS gel showing recombinant Xenopus (X), mouse (M) and human (H) pol γB subunits. 4.7 pmol of each protein was loaded on the gel, corresponding to ~230 ng of each polypeptide. The sizes in kDa of molecular mass markers run in parallel are indicated on the left. (B) Primer extension assay on oligo(dT)-primed poly(dA). Reactions were carried out as described in Materials and Methods using 40 fmol of pol γA and 240 fmol of pol γB per reaction. Lane 1, no protein; lanes 2–5 correspond to reactions carried out with pol γA alone, A + Xenopus B, A + mouse B and A + human B, respectively, incubated for 1 min; lanes 6–9 are as 2–5 but incubated for 2 min; lanes 10–13 correspond to reactions incubated for 5 min. The sizes in nucleotides of MspI restriction fragments of pUC 18 are indicated on the left.
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