Abstract
The proteolytic removal of about 60 amino acids from the COOH terminus of the bacteriophage T4 helix-destabilizing protein (gene 32 protein) produces 32*I, a 27,000-dalton fragment which still binds tightly and cooperatively to single-stranded DNA. The substitution of 32*I protein for intact 32 protein in the seven-protein T4 replication complex results in dramatic changes in some of the reactions catalyzed by this in vitro DNA replication system, while leaving others largely unperturbed. 1. Like intact 32 protein, the 32*I protein promotes DNA synthesis by the DNA polymerase when the T4 polymerase accessory proteins (gene 44/62 and 45 proteins) are also present. The host helix-destabilizing protein (Escherichia coli ssb protein) cannot replace the 32I protein for this synthesis. 2. Unlike intact 32 protein, 32*I protein strongly inhibits DNA synthesis catalyzed by the T4 DNA polymerase alone on a primed single-stranded DNA template. 3. Unlike intact 32 protein, the 32*I protein strongly inhibits RNA primer synthesis catalyzed by the T4 gene 41 and 61 proteins and also reduces the efficiency of RNA primer utilization. As a result, de novo DNA chain starts are blocked completely in the complete T4 replication system, and no lagging strand DNA synthesis occurs. 4. The 32*I protein does not bind to either the T4 DNA polymerase or to the T4 gene 61 protein in the absence of DNA; these associations (detected with intact 32 protein) would therefore appear to be essential for the normal control of 32 protein activity, and to account at least in part for observations 2 and 3, above. We propose that the COOH-terminal domain of intact 32 protein functions to guide its interactions with the T4 DNA polymerase and the T4 gene 61 RNA-priming protein. When this domain is removed, as in 32*I protein, the helix destabilization induced by the protein is controlled inadequately, so that polymerizing enzymes tend to be displaced from the growing 3'-OH end of a polynucleotide chain and are thereby inhibited. Eukaryotic helix-destabilizing proteins may also have similar functional domains essential for the control of their activities.
Highlights
PROTEOLYTIC REMOVAL OF THE CARBOXYL TERMINUS OF THE T4 GENE 32 HELIX-DESTABILIZING PROTEIN ALTERS THE T4 IN VITRO REPLICATION COMPLEX
(detected with intact 32 protein) would appear to be essential for the normal control of 32 protein activity, and to account at least in part for observations 2) and 3), above, We propose that the carboxyl-terminal domain of intact 32 protein functions to guide its interactions with the T4 DNA polymerase and the T4 gene 61 RNA priming protein, When this domain is removed, as in 32'~I protein, the helix-destabilization induced the protein is inadequately c.ontrolled, so that polymerizing enzymes tend to be displaced from the grm"ing 3' OH end of a polynucleotide c.hain and are thereby inhibited, Tn vit.ro DNA s is is efficien by a mul complex of seven purified proteins encoded bacter1o~
Is double·-stranded, circular PM2 DNA. which has been nicked once per circle, such a initiation is limited to one event per molecule, tion s in a ling circlet! mode to generate single~stranded tails which are much longer than unit length [5], The addition of the RNA pr proteins (41 protein and 61 protein) to this five~protein reaction stimulates incorporation approximately t1:vO fold, even without the ribonucleotide substrates rGTP, rCTP, and rUTP, This primer~'independent stimulation requires the presence of 41 protein and its rGTP hydrolys:Ls [2], It arises from an acceleration of the rate at which a fraction of the forks
Summary
Seven highly purified T4 bacteriophage encoded proteins constitute the presentLl?:. ~~!r~_ T4 DNA replication system: the helix-destabilizing protetn (gene 32 protein), the DNA polymerase (the 43 gene protein), the polymerase accessory proteins (45 protein plus a tight complex of 4!+ and 62 proteins), and the RNA priming proteins (41 protein and 61 protein). Pri.ming, in the T4 in ~_i:.!=I:~_ replica ti.on system, all seven purified replication proteins and ribonucleotide (and deoxyribonucleotide) substrates are essential to observe replication on an fd DNA e, The eventual product of the reaction is the same as that obtained with a singly nicked duplex circular template - a double-stranded circle with a long tail containing alternating singlestranded and double-stranded [38]. This inhibition is competitive, as shown by the gradual increase in synthesis as the ratio of 32 to 32*1 protein in the reaction mix is increased (Fig. 5), With an equal concentration of the two forms of 32 protein present, 80% inhibition is seen, and the block is comp1e relieved only when the ratio of 32 to 32*1 reaches 4:1, The observed inhibition of de novo initiations could arise because. This observation 18 relevant to the fact that T4 DNA takes place in a cell which contains the E. coli H-D protein
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