Cloning and expression of human pol γB
Primers HGBNDE (5′-ACTTCGGAGACATATGCGCTCT-CGTGTAGCCG-3′) and HGBNOT (5′-CAAATATAGCGG-CCGCTACATTCTTAGCTGATGAT-3′) were used to amplify a 1.5 kb PCR product from a HeLa Marathon-ready cDNA (Clontech), using Pfu
Turbo DNA polymerase (Stratagene). This product was cloned as an Nde
I fragment in the vector pET22b(+) (Novagen) to generate clone pJAC39. Proteins were expressed in Escherichia coli
strain BL21(DE3) after induction with 1 mM IPTG. Sequencing of this clone revealed the presence of several differences with the previously published sequence, including two frame shifts. Primer HGB1 (5′-TCATATGGATGCGGGGCAGCCGGAGC-3′), corresponding to sequence DAGQPE preceded by a methionine codon, was used in combination with the HGBNOT primer to clone by PCR a cDNA encoding the putative mature protein (clone pJAC44). For expression of proteins in insect cells, the Nde
I insert was directly transferred into a pFastBacI baculovirus expression vector (Life Technologies) modified as previously described (11
) to allow expression in insect cells of C-terminal his-tagged proteins. The human sequence has been deposited in GenBank under accession number AF177201.
Cloning and expression of mouse pol γB
Primer PMGB1 (5′-CTGCAGGTGCTGGCTGTCCG-3′) was designed from EST clone AA499566 and used in PCR together with primer MGBNOT (5′-GGCGGCCGCCACATTGCTAGCTGACGCTAG-3′) using a mouse Marathon-ready cDNA (Clontech) as template and Pfu DNA polymerase (Stratagene). We amplified two fragments of 1.2 and 1.3 kb that were cloned into the EcoRV site of plasmid pBluescript II KS(+) to generate clones pJAC52 (short product) and pJAC54 (large product). Sequencing revealed a 125 bp fragment missing in pJAC52 with respect to pJAC54. For bacterial expression, PCR on pJAC54 DNA with primer MGBNDE (5′-CGCAGGGC-CTGCCATATGTGGCTGTCCGGGTACGCG-3′) and primer MGBNOT was used to produce an NdeI–NotI fragment to express the putative mature protein in bacteria using the pET22b(+) vector. The mouse sequence has been deposited in GenBank under accession number AF177202.
Purification of his-tagged proteins
Bacteria expressing the recombinant protein were washed in a buffer containing 50 mM HEPES (pH 7.5), 0.2 M sucrose, 1 mM EDTA, 1 mM PMSF, 1 µM pepstatin and 7 mM β-mercaptoethanol, and sonicated in lysis buffer (50 mM sodium phosphate, pH 8, 300 mM NaCl, 20% glycerol, 1 mM β-mercaptoethanol, 0.5 mM benzamidine–HCl, 1 µM pepstatin, 5 µg/ml leupeptin and 0.2 mM PMSF). Insoluble proteins were solubilized in the same buffer containing 8 M urea without glycerol. Insect cells expressing recombinant protein were lysed by sonication in binding buffer (50 mM sodium phosphate, pH 8, 300 mM NaCl, 10% glycerol, 3 mM β-mercaptoethanol and 0.2 mM PMSF).
For affinity purification on Ni-NTA columns, imidazole was added to 10 mM final concentration to the cell lysate containing soluble proteins. Both bacterial and insect cell lysates were precleared by centrifugation at 23 500 g for 10 min. The cleared lysates were incubated for 1 h with Ni-NTA beads (Qiagen) pre-washed in binding buffer containing 20 mM imidazole (W20). Proteins from insect cells were then washed three times with W20 and proteins from bacteria were washed in W60 (binding buffer containing 60 mM imidazole). Elution was carried out with EB250 (binding buffer containing 250 mM imidazole). Purified protein was dialyzed against a buffer containing 50 mM sodium phosphate, pH 8, 300 mM NaCl, 50% glycerol, 2 mM β-mercaptoethanol and 0.2 mM PMSF, and stored at –80°C in aliquots after freezing in liquid nitrogen. To prepare protein to be used as antigen, bacterially expressed protein was purified from the urea-soluble fraction as described above but using buffers containing 8 M urea. Purified protein was dialyzed against PBS and used to inoculate a rabbit.
Partial purification of DNA pol γ from HeLa cells
HeLa cells were grown in 12 l of DMEM to a cell density of 7 × 105 cells/ml. Cells were harvested by centrifugation and washed in 240 ml TD buffer (135 mM NaCl, 5 mM KCl, 25 mM Tris–Cl, pH 7.6). After centrifugation as above, cells were resuspended in 210 ml of CaRSB buffer (10 mM NaCl, 1.5 mM CaCl2, 10 mM Tris, pH 7.5) and allowed to swell for 10 min on ice. Cells were then homogenized with a glass Dounce homogenizer and two-thirds volume of 2.5× MS buffer was added (1× MS is 210 mM mannitol, 70 mM sucrose, 5 mM EDTA, 5 mM Tris, pH 7.6). Nuclei were pelleted by centrifugation at 1500 g for 5 min, twice. A mitochondrial pellet was obtained by centrifugation at 27 000 g for 15 min, mitochondria were resuspended in MS buffer and then centrifuged again. Mitochondrial lysis was achieved by homogenization in MS buffer after addition of KCl to 250 mM and Triton X-100 to 0.5%. The mitochondrial lysate was clarified by centrifugation at 50 000 r.p.m. in a Beckman 70Ti rotor for 1 h and stored at –80°C.
Purification of pol γ was carried out by chromatography of the lysate through DEAE-Sephacel at 250 mM KCl in buffer Z (20 mM Tris pH 8.4, 1 mM DTT, 1 mM EDTA, 10% glycerol, 0.2 mM PMSF, 10 mM benzamidine–HCl, 1 µM pepstatin, 1 µg/ml aprotinin, 1 µg/ml leupeptin). The flow through was dialyzed against buffer Z to a KCl concentration below 50 mM, loaded onto a SP-Sepharose column and proteins were eluted with a KCl gradient from 50 to 750 mM in the same buffer. The peak fractions were collected, pooled, diluted to a KCl concentration of 200 mM and loaded onto a Heparin Hi Trap column. Proteins were eluted with a KCl gradient from 200 mM to 1 M. Pol γ was detected by assay for reverse transcriptase activity on poly(rA):oligo(dT), as described previously (2
Generation of a rabbit antiserum against human pol γA
The polymerase domain of human pol γA was expressed in bacteria as a C-terminal his-tagged protein from an expression vector constructed as follows. An NdeI-containing primer was made in such a way that the ATG in the NdeI site matched the ATG in the sequence MDQEDL (between the exonuclease and the polymerase domains in the protein). Another primer was made containing a NotI site corresponding to the stop codon of the cDNA. PCR with these two primers on a HeLa Marathon-ready cDNA (Clontech) amplified a 2180 bp fragment that was then digested with NdeI and NotI and cloned into pET22B(+) (a three piece ligation was done due to the existence of an internal NdeI site in the amplified DNA). The 81.7 kDa protein was expressed in E.coli and purified from urea-solubilized inclusion bodies by affinity chromatography on Ni-NTA under denaturing conditions (8 M urea). A rabbit was immunized using standard protocols.
Human pol γB deletion mutants
To generate the different deletion mutants described in this work, NdeI–NotI fragments produced by PCR were cloned into pET22b(+) to produce C-terminal his-tagged proteins as described for the wild-type protein. N-terminal deletions were made by PCR with Pfu turbo DNA polymerase on pJAC44 DNA using the T7 terminator primer in combination with each of the following primers: HGBDN1 (5′-GAAGGCATTTCCTAC-ATATGAGCAAGCAGCAGCTTA-3′) and HGBDN2 (5′-GACCCTTGGGCGTACATATGCGGAAGAACCTGGC-3′). C-terminal deletions were produced using the same approach with the T7 promoter primer in combination with each of the following: HGBDC1 (5′-TCATTGTGGTGTCTGCGGCCG-CCAGATGTATTAATCCA-3′), HGBDC2 (5′-AACAAGGGT-GAAGTGCGGCCGCCTTTCTATGAAGATTT-3′), HGBDC3 (5′-CCACATTTTTTCGTGCGGCCGCGCCATGTAATTTAG-AC-3′). These PCR products were digested with NdeI and NotI and cloned into pET22b(+). Internal deletion HGBΔI1 was produced by elimination of a 240 bp SmaI fragment and religation. HGBΔI2 was generated by replacing the NdeI–HindIII fragment from pJAC 44 with an NdeI–HindIII fragment from a PCR product obtained with the T7 promoter primer and primer HGBHIND (5′-AATCTGAGAAGCTTCATAAGGTAGCC-TCTTGTT-3′). To generate HGBΔI3, a PCR product was obtained with the T7 promoter primer and primer HGBECO1 (5′-ACTGAATTCGGGTAGCAAAGGGCCTG-3′), a second PCR product was generated with primer HGBECO2 (5′-GTT-GAATTCGAAACTCTACGCGAAAT-3′) and the T7 terminator primer. NdeI–EcoRI and EcoRI–NotI fragments from these two PCR products were ligated together with NdeI–NotI digested pET22b(+). Restriction analysis and sequencing confirmed that the clones were correct. Protein expression in bacteria and purification by affinity chromatography on Ni-NTA was achieved as described above.
Pol γ activity assays
Pol γ assays and electrophoretic mobility shift assays (EMSA) were carried out as described (11
). For the EMSA assays shown in Figure , we used an internally labeled [α32
P]dCTP 45mer (5′-ATCCAACCTCGCGTCGTATCGAATCGGATCA-GATCGGGTCGTC
AA-3′, labeled C is underlined) annealed to a 26mer (5′-TTGACGACCCGATCTGATCCGATTCG-3′), which leaves a 19-nt single-stranded region.
Figure 4 Deletion analysis of human pol γ B. (A) Diagram showing the locations of deletions within human pol γB in comparison to the full-length mature protein (HGB). Black rectangles indicate the regions that align with conserved motifs 1–3 (more ...)