Biotin Tagging Deletion Analysis of Domain Limits Involved in Protein-Macromolecular Interactions

Abstract

The subunit dimerizes DNA polymerase III via interaction with the ␣ subunit, allowing DNA polymerase III holoenzyme to synthesize both leading and lagging strands simultaneously at the DNA replication fork. Here, we report a general method to map the limits of domains required for heterologous protein-protein interactions using surface plasmon resonance. The method employs fusion of a short biotinylation sequence at either the NH 2 or COOH terminus of the protein to be immobilized on streptavidin-derivatized biosensor chips. Inclusion of a hexahistidine sequence permits rapid purification and separation of the fusion protein from the endogenous Escherichia coli biotin carboxyl carrier protein. Ten deletions of the ␣ subunit were constructed and purified by Ni 2؉ -nitrilotriacetic acid chromatography and, when required, monomeric avidin chromatography. Each ␣ deletion protein was captured by streptavidin immobilized on a Pharmacia Biosensor BIAcore chip, and the binding activity of each ␣ deletion was analyzed using surface plasmon resonance. The subunit bound very tightly to a full-length amino-terminal fusion of the biotinylation sequence with ␣ (K D ϳ 70 pM). Four additional NH 2 -terminal ␣ deletion proteins (60, 240, 360, and 542 residues deleted) retained strong binding activity to the subunit (K D ‫؍‬ 0.19 -0.39 nM), whereas deletion of 705 residues or more from the NH 2 terminus of the ␣ subunit abolished binding activity. Full-length ␣ that contained a carboxyl-terminal fusion with the biotinylation sequence bound strongly (K D ‫؍‬ 0.37 nM). However, deletion of 48 amino acids from the COOH terminus totally eliminated binding. These results indicate that the COOH-terminal half of the ␣ subunit is involved in interaction. DNA polymerase III holoenzyme (holoenzyme)1 is the major DNA polymerase responsible for Escherichia coli chromosomal DNA synthesis. Holoenzyme consists of 10 individual subunits: ␣, ⑀, , , ␥, ␦, ␦Ј, , , and ␤ (McHenry, 1988;Kornberg, 1988), all of which act cooperatively in the coordinate and processive synthesis of leading and lagging strands (Wu et al., 1992a(Wu et al., , 1992bFay et al., 1982). Each subunit is encoded by a separate gene on the chromosome, except for and ␥, which are both expressed from dnaX. The subunit (71 kDa) is the full-length product of the dnaX gene; the ␥ subunit (47 kDa) is synthesized by a Ϫ1 translational frameshift and comprises the NH 2 -terminal two-thirds of the subunit (McHenry et al., 1989;Tsuchihashi and Kornberg, 1990;Blinkowa and Walker, 1990;Flower and McHenry, 1990).Polymerase III core (pol III) is composed of three different subunits: ␣, ⑀, and (McHenry and Crow, 1979). The ␣ subunit (dnaE product), a 130-kDa polypeptide, has intrinsic DNA polymerase activity (Welch and McHenry, 1982;Maki et al., 1985), the ⑀ subunit (dnaQ product) contains 3Ј 3 5Ј exonuclease activity for proofreading (Scheuermann and Echols, 1984), and the function of the subunit (holE product) is unknown (Carter et al., 1993;Studwell-Vaughan et al., 1993;Slater et al., 1994). ...

A peptide switch regulates DNA polymerase processivity

“…4, demonstrates that wild-type core forms a stable complex with , but core ⌬20 does not. This finding is consistent with an earlier study (20) showing that a mutant ␣ subunit lacking 48 C-terminal residues could not form a stable complex with . To measure the affinity of to the ␣ C-tail peptide, we used the 24-kDa C-terminal section of ( c ), which contains the domain responsible for binding ␣.…”

“…To determine the precise location of the contact site between ␤ and the ␣ polymerase subunit of the heterotrimeric Pol III core (␣ ), we synthesized an overlapping set of peptides (20-mers) that spanned the region of ␣ (812-991) previously reported to bind ␤ (19,20). Peptides were immobilized via N-terminal biotin to streptavidin-coated microtiter plates and then assayed for the ability to retain [ 32 P]␤ in the well.…”

Essential roles for imuA ′- and imuB -encoded accessory factors in DnaE2-dependent mutagenesis in Mycobacterium tuberculosis

“…Unlike Y-family polymerases whose structures are adapted to specialist lesion bypass (20), sequence analysis reveals few clues to DnaE2 function. All major DNA polymerase IIIα structural/functional domains (23,24) are readily identified in DnaE2, except for the very C-terminal region which in E. coli has been implicated in the interaction of α with the clamploader subunit, τ (28,29). A strong α-τ interaction enables simultaneous leading and lagging-strand synthesis by the DNA polymerase III holoenzyme (13), and the absence of this region in DnaE2 and all other nonessential dnaE-type α-subunits (Fig.…”

DNA Polymerase III Holoenzyme from Thermus thermophilus Identification, Expression, Purification of Components, and Use to Reconstitute a Processive Replicase

“…To construct pA1-NB-AvrII, pDRK-N(M), a plasmid designed for expression of proteins with an amino-terminal tag was used as the starting plasmid (24). The amino-terminal tag is composed of a 30-amino acid peptide that is biotinylated in vivo and contains a hexahistidine sequence.…”

“…Pol III consists of ␣, the catalytic polymerase subunit associated with the ⑀ 3Ј 3 5Ј exonuclease, and (22). Pol III gains its special replicative properties by its ability to associate with ␤ and through interactions enabled by sequences located in the carboxyl-terminal third of the ␣ subunit (23)(24)(25)(26).…”