Bacteriophage T4 gene 41 protein, required for the synthesis of RNA primers, is also a DNA helicase.

M Venkatesan,L L Silver,N G Nossal

doi:10.1016/s0021-9258(18)33731-1

Abstract

Bacteriophage T4 gene 41 protein is one of the two phage proteins previously shown to be required for the synthesis of the pentaribonucleotide primers which initiate the synthesis of new chains in the T4 DNA replication system. We now show that a DNA helicase activity which can unwind short fragments annealed to complementary single-stranded DNA copurifies with the gene 41 priming protein. T4 gene 41 is essential for both the priming and helicase activities, since both are absent after infection by T4 phage with an amber mutation in gene 41. A complete gene 41 product is also required for two other activities previously found in purified preparations of the priming activity: a single-stranded DNA-dependent GTPase (ATPase) and an activity which stimulates strand displacement synthesis catalyzed by T4 DNA polymerase, the T4 gene 44/62 and 45 polymerase accessory proteins, and the T4 gene 32 helix-destabilizing protein (five-protein reaction). The 41 protein helicase requires a single-stranded DNA region adjoining the duplex region and begins unwinding at the 3' terminus of the fragment. There is a sigmoidal dependence on both nucleotide (rGTP, rATP) and protein concentration for this reaction. 41 Protein helicase activity is stimulated by our purest preparation of the T4 gene 61 priming protein, and by the T4 gene 44/62 and 45 polymerase accessory proteins. The direction of unwinding is consistent with the idea that 41 protein facilitates DNA synthesis on duplex templates by destabilizing the helix as it moves 5' to 3' on the displaced strand.

Highlights

Thesis of the pentaribonucleotide primers which initiadteependent reaction and function at least in part by decreasing the synthesisof new chains in theT4 DNA replication the dissociation of the polymerase from the growing chain system
DNA-dependenGt TPase(ATPasea)ndan activity accessory proteins, and the gene 32 helix-destabilizingprotein which stimulates strand displacement synthesis cata- [1,2,10]. synthesis at a nick proceeds in the absence lyzed by T4 DNA polymerase, theT4 gene 44/62 and 45 polymeraseaccessoryproteins,andthe T4 gene 32 of the ribonucleotide triphosphates and 61 and 41 proteins which are required for lagging strand synthesis (1, 2, la), 41 helix-destabilizing protein. protein doesincrease the rateof strand displacement synthesis
Using a singly nicked circular template, we show preparation of theT4 gene 61 priming protein, and by that 41 protein markedly increases the rate at which some the T4 gene 44/62 and 45 polymerase accessory pro- chains are elongated by the other five T4 proteins

Summary

Introduction

Thesis of the pentaribonucleotide primers which initiadteependent reaction and function at least in part by decreasing the synthesisof new chains in theT4 DNA replication the dissociation of the polymerase from the growing chain system. Using a singly nicked circular template, we show preparation of theT4 gene 61 priming protein, and by that 41 protein markedly increases the rate at which some the T4 gene 44/62 and 45 polymerase accessory pro- chains are elongated by the other five T4 proteins. The gene 41 and 61 proteins together catalyze the synthesis of the pentaribonucleotide primers, with the predominant sequence pppAC(N)a,which are used to initiate the synthesis ofnew DNA chains in vitro [3,4,5] and are found at the termini of Okazaki fragments in vivo [6] These primers are elongated on single-stranded templates by the polymerase and the gene 44/62 and gene 45 polymerase accessory proteins [1, 2].The

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Conclusion