[43] Purification of bacteriophage T4 DNA replication proteins

Nossal, Nancy G.; Hinton, Deborah M.; Hobbs, Lisa J.; Spacciapoli, Peter

doi:10.1016/0076-6879(95)62045-1

Cited by 39 publications

(40 citation statements)

References 43 publications

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“…Synthesis was then initiated by addition of a mixture of T4 DNA polymerase (30 nM), 61 primase (64 nM), and 41 helicase (328 nM monomer). At the times indicated, aliquots of the reaction mixtures were mixed with an equal volume of 0.2 M EDTA to stop the synthesis, and the products were analyzed by 0.6% alkaline agarose gel electrophoresis (33) and trichloroacetic acid precipitation (29).…”

Section: јmentioning

confidence: 99%

Mutations of Bacteriophage T4 59 Helicase Loader Defective in Binding Fork DNA and in Interactions with T4 32 Single-stranded DNA-binding Protein

Jones

Green

Stephens

et al. 2004

Journal of Biological Chemistry

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Bacteriophage T4 gene 59 protein greatly stimulates the loading of the T4 gene 41 helicase in vitro and is required for recombination and recombination-dependent DNA replication in vivo. 59 protein binds preferentially to forked DNA and interacts directly with the T4 41 helicase and gene 32 single-stranded DNA-binding protein. The helicase loader is an almost completely ␣-helical, two-domain protein, whose N-terminal domain has strong structural similarity to the DNA-binding domains of high mobility group proteins. We have previously speculated that this high mobility group-like region may bind the duplex ahead of the fork, with the C-terminal domain providing separate binding sites for the fork arms and at least part of the docking area for the helicase and 32 protein. Here, we characterize several mutants of 59 protein in an initial effort to test this model. We find that the I87A mutation, at the position where the fork arms would separate in the model, is defective in binding fork DNA. As a consequence, it is defective in stimulating both unwinding by the helicase and replication by the T4 system. 59 protein with a deletion of the two C-terminal residues, Lys 216 and Tyr 217 , binds fork DNA normally. In contrast to the wild type, the deletion protein fails to promote binding of 32 protein on short fork DNA. However, it binds 32 protein in the absence of DNA. The deletion is also somewhat defective in stimulating unwinding of fork DNA by the helicase and replication by the T4 system. We suggest that the absence of the two terminal residues may alter the configuration of the lagging strand fork arm on the surface of the C-terminal domain, so that it is a poorer docking site for the helicase and 32 protein.Bacteriophage T4 DNA replication begins by synthesis from one of several origins in the early stage of infection, but replication from forks created on recombination intermediates becomes the predominant replication initiation mechanism at later times (1, 2). Recombination-dependent replication is made possible by the terminally redundant and circularly permuted arrangement of the 168-kb linear T4 DNA genome. Thus, the end of one molecule can invade the homologous internal region of another molecule. Phage T4 encodes the replication proteins that are needed for both modes of replication (reviewed in Refs. 3 and 4).In the T4 replication system, gene 41 helicase unwinds the duplex ahead of the leading strand T4 DNA polymerase and associates with the gene 61 primase to enable it to make the pentamer primers that initiate each lagging strand fragment. In vitro, the helicase by itself loads slowly on replication forks, but its loading is greatly stimulated by the gene 59 helicase loading protein (5). In vivo, the helicase loading protein is essential for recombination and recombination-dependent replication (1, 2). Both 41 helicase and its 59 loader are needed for polar branch migration on joint molecules formed by the T4 UvsX recombinase and gene 32 single-stranded DNA-binding protein (6), and the helicase, 59 l...

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Section: јmentioning

confidence: 99%

Mutations of Bacteriophage T4 59 Helicase Loader Defective in Binding Fork DNA and in Interactions with T4 32 Single-stranded DNA-binding Protein

Jones

Green

Stephens

et al. 2004

Journal of Biological Chemistry

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“…1 and 3, can contribute to DNA repair and explain ''homing'' of intron DNA (15,18,19). Most of the proteins involved in these pathways have been biochemically characterized in exquisite detail (2,12,(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35).…”

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confidence: 99%

Two recombination-dependent DNA replication pathways of bacteriophage T4, and their roles in mutagenesis and horizontal gene transfer

Mosig

Gewin

Luder

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

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. In wild-type T4, timing of these pathways is integrated with the developmental program and related to transcription and packaging of DNA. In primase mutants, which are defective in origin-dependent lagging-strand DNA synthesis, the late pathway can bypass the lack of primers for lagging-strand DNA synthesis. The exquisitely regulated synthesis of endo VII, and of two proteins from its gene, explains the delay of recombination-dependent DNA replication in primase (as well as topoisomerase) mutants, and the temperature-dependence of the delay. Other proteins (e.g., the singlestranded DNA binding protein and the products of genes 46 and 47) are important in all recombination pathways, but they interact differently with other proteins in different pathways. These homologous recombination pathways contribute to evolution because they facilitate acquisition of any foreign DNA with limited sequence homology during horizontal gene transfer, without requiring transposition or site-specific recombination functions. Partial heteroduplex repair can generate what appears to be multiple mutations from a single recombinational intermediate. The resulting sequence divergence generates barriers to formation of viable recombinants. The multiple sequence changes can also lead to erroneous estimates in phylogenetic analyses.The purpose of looking back is not, of course, merely to obtain satisfaction from reflecting on past triumphs; rather, it is to discover as many clues as possible to the likely developments of the future.Glenn T. Seaborg T he tight interrelationship between homologous recombination and DNA replication was first evident in T4 and the related T-even phages. Because DNA of T4 and its host E. coli differ in base composition and modifications and because the host DNA is rapidly degraded after phage infection, molecular aspects of T4 replication and recombination could be readily investigated by biochemical, biophysical, and genetic methods. Early characterization of mutations in most essential genes (1) and the almost complete dependence of replication and recombination on phage-encoded proteins (2) allowed analyses of recombination and replication proteins, as well as ''reality checks'' of results obtained with genetic and biochemical methods (3). The following idiosyncrasies of T4 chromosomes revealed the importance of DNA ends and recombination-dependent DNA replication. Ends of T4 chromosomes are cut during packaging from branched concatemers, which are generated by recombination-dependent replication. A ''headful mechanism'' packages a complete genome and Ϸ3% DNA repeated at each end as ''terminal redundancy,'' thereby generating the random circular permutation of chromosomal ends (4). Some smaller T4 particles, formed because of assembly errors, package incomplete genomes whose ends are also randomly circularly permuted (5). Multifactor crosses revealed stimulation of recombination by their DNA ends, regardless of map positions (5, 6). Moreover, different segregation patterns of alleles in patch vs. splice re...

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“…The proteins were first purified by chromatography on DEAE-Sepharose and ssDNA-cellulose as described (15). T4 gp32mms from the DEAE-Sepharose column binds efficiently to the ssDNA-cellulose column only when the rate of loading is about 5 ml/h per cm 2 of the column.…”

Section: Methodsmentioning

confidence: 99%

“…Protein Purifications-Overproduction and purification of T4 gp43, gp44/62, gp45, gp59 and gp61 were performed as described (13) based on earlier protocols (15). T4 gp41uvs79 was overproduced in Bl21(DE3)/ plysS cells bearing the plasmid coding for the mutant gp41.…”

Section: Methodsmentioning

confidence: 99%

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Properties of Bacteriophage T4 Proteins Deficient in Replication Repair

Kadyrov

Drake

2003

Journal of Biological Chemistry

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An epistasis group of mutations engendering increased sensitivity to diverse DNA-damaging agents was described previously in bacteriophage T4. These mutations are alleles of genes 32 and 41, which, respectively, encode a single-stranded DNA-binding protein (gp32) and the replicative DNA helicase (gp41). The mechanism by which the lethality of DNA damage is mitigated is unknown but seems not to involve the direct reversal of damage, excision repair, conventional recombination repair, or translesion synthesis. Here we explore the hypothesis that the mechanism involves a switch in DNA primer extension from the cognate template to an alternative template, the just-synthesized daughter strand of the other parental strand. The activities of the mutant proteins are reduced about 2-fold (for gp32) or 4-fold (for gp41) in replication complexes catalyzing coordinated synthesis of leading and lagging strands, in binding single-stranded DNA, promoting DNA annealing, and promoting branch migration. In striking contrast, the mutant proteins are strongly impaired in promoting template switching, thus supporting the hypothesis of survival by template switching.Four general mechanisms that either repair or circumvent potentially lethal damage to DNA have been extensively investigated. These are (i) direct reversal of the damage, as in photoreactivation or dealkylation, (ii) damage excision followed by resynthesis templated by the complementary strand, as in base excision repair and nucleotide excision repair, (iii) recombinational repair, in which the damaged DNA obtains the requisite sequence information from another chromosome or from the sister chromosome in a way that depends on a recombinase and a Holliday-junction resolvase, and (iv) translesion synthesis, which is frequently mutagenic. Strong hints of a fifth mechanism have accumulated over time, but a combined genetic and enzymological demonstration of such a mechanism has not been available. These hints accumulated in two parallel lines of investigation, one using eukaryotic cells and the other using bacteriophage T4. Both lines point toward a repair mechanism based on a template-switching process operating directly at the replication fork.In a classic model of the fifth mechanism (1), DNA replication on one parental template strand is blocked by a lesion while replication on the other parental strand continues briefly, whereupon strand displacement and branch migration re-associate the two daughter strands into a short duplex (Fig. 1). The blocked daughter strand then continues replication on the alternative template, and then the structure reforms a conventional replication fork with the lesion having been bypassed. This model was supported by experiments in which bromodeoxyuridine was used as a "heavy" label and tritiated thymidine as a "light" label during the replication of DNA in UV-irradiated mammalian cells: a small amount of tritiated DNA of "heavy-heavy" density was observed in DNA from irradiated cells, and this was chased into "heavy-light" DNA. In addition...

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[43] Purification of bacteriophage T4 DNA replication proteins

Cited by 39 publications

References 43 publications

Mutations of Bacteriophage T4 59 Helicase Loader Defective in Binding Fork DNA and in Interactions with T4 32 Single-stranded DNA-binding Protein

Mutations of Bacteriophage T4 59 Helicase Loader Defective in Binding Fork DNA and in Interactions with T4 32 Single-stranded DNA-binding Protein

Two recombination-dependent DNA replication pathways of bacteriophage T4, and their roles in mutagenesis and horizontal gene transfer

Properties of Bacteriophage T4 Proteins Deficient in Replication Repair

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