T4 Phage Gene 32 Protein as a Candidate Organizing Factor for the Deoxyribonucleoside Triphosphate Synthetase Complex

Abstract

After T4 bacteriophage infection of Escherichia coli, the enzymes of deoxyribonucleoside triphosphate biosynthesis form a multienzyme complex that we call T4 deoxyribonucleoside triphosphate (dNTP) synthetase. At least eight phage-coded enzymes and two enzymes of host origin are found in this 1.5-mDa complex. The complex may shuttle dNTPs to DNA replication sites, because replication draws from small pools, which are probably highly localized. Several specific protein-protein contacts within the complex are described in this paper. We have studied protein-protein interactions in the complex by immobilizing individual enzymes and identifying radiolabeled T4 proteins that are retained by columns of these respective affinity ligands. Elsewhere we have described interactions involving three T4 enzymes found in the complex. In this paper we describe similar analysis of five more proteins: dihydrofolate reductase, dCTPase-dUTPase, deoxyribonucleoside monophosphokinase, ribonucleotide reductase, and E. coli nucleoside diphosphokinase,. All eight proteins analyzed to date retain single-strand DNA-binding protein (gp32), the product of T4 gene 32. At least one T4 protein, thymidylate synthase, binds directly to gp32, as shown by affinity chromatographic analysis of the two purified proteins. Among its several roles, gp32 stabilizes single-strand template DNA ahead of a replicating DNA polymerase. Our data suggest a model in which dNTP synthetase complexes, probably more than one per growing DNA chain, are drawn to replication forks via their affinity for gp32 and hence are localized so as to produce dNTPs at their sites of utilization, immediately ahead of growing DNA 3' termini.

Molecular and Functional Interactions between Escherichia coli Nucleoside-diphosphate Kinase and the Uracil-DNA Glycosylase Ung

“…One such region may be the so called "Kpn loops" located on the north and south poles of the NDP kinase multimer (2), to which modulating functions have already been assigned both in the classical case of the killer-of-prune (kpn) synthetic lethal interaction between PRUNE, a phosphodiesterase (36), and AWD, the Drosophila NDK (8,37), and also in a case of NM23-H2 human NDK (20). From a physiological perspective, this interaction is not surprising, as the presence of both enzymes has been previously implicated in association with a DNA replication complex (Ndk with a T4 bacteriophage replication complex (22) and Ung in the proximity of other components of the DNA repair machinery of the E. coli chromosome (38)). From the genetic viewpoint, however, it is a puzzle, because, although mutant E. coli cells lacking either Ndk or Ung have mutator phenotypes, the mutations they accumulate differ (27,39).…”

“…coli Ndk is also multifunctional; it can directly interact with a half dozen other proteins involved in DNA metabolism (22,23) and has been found in a DNA replication complex (23),…”

“…coli Ndk is also multifunctional; it can directly interact with a half dozen other proteins involved in DNA metabolism (22,23) and has been found in a DNA replication complex (23), * This work was supported by Grant ESO6070 (to G. P. P.) from the National Institutes of Health and by Grant RO1CA076496 (to E. H. P.) from NCI, National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges.…”

Crystal structure of deoxycytidylate hydroxymethylase from bacteriophage T4, a component of the deoxyribonucleoside triphosphate-synthesizing complex

“…A relatively small interface area between T4 CH and T4 TS in the proposed complex suggests a weak binding of the two enzymes, as the binding energy correlates well with the buried surface area in the protein-protein interface (Horton and Lewis, 1992). T4 TS was also shown to interact with several T4 proteins including dCMP deaminase, DHFR and singlestranded DNA binding protein (gp32) (McGaughey et al, 1996;Wheeler et al, 1996), as well as CH. As a result, the interaction between T4 CH and T4 TS needs to be limited in its extent and sufficient surface areas of T4 TS outside the interface with T4 CH should be available for interactions with other proteins.…”

Domain Effects on the DNA-interactive Properties of Bacteriophage T4 Gene 32 Protein

“…The C-terminal (A) domain has a major role in gene 32 protein interaction with other T4 proteins (10,11), a property that is the basis of viewing this protein as a "candidate organizing factor" for protein-protein interactions (12). This domain has another very interesting property: it modulates the ability of the protein to lower the melting temperature of natural double-stranded DNA helices.…”