Origins of phage T4 DNA replication as revealed by hybridization to cloned genes.

Two physical origins ofT4 DNA replication were determined by hybridization of viral DNA prepared 2.5 min after infection to a display oftotal T4 DNA. This is the earliest time after T4 infection ofEacherichia coli at 37C that labeled and hybridizable DNA can be detected. The two origins, separated by about 25 kdlobases, were identified and localized in the early region of the T4 map. One of them is located in a 5.6-kilobase EcoRI fragment containinggenes . The other is located between genes rl and e in a 1.9-kllobase EcoRI fragment. Both of these T4 fragments have been cloned and their interactions with the host cell are discussed.The complex process of DNA replication involves specific initiation at defined locations of DNA called origins and subsequent chain elongation and fork movement. The T4 system, with its well-defined genetics, has been most valuable in understanding the enzymology and mechanism ofchain elongation and fork movement, which is catalyzed by a multienzyme complex (1). However, little is known about the process by which T4 DNA replication is initiated. We The fact that different groups ofworkers, using different culturing and infecting conditions, have apparently identified different regions ofthe T4 genome as origins suggests that multiple but specific sites may all be used. It is therefore of importance to characterize each ofthese origins in as great detail as possible in the hope that some general scheme will emerge leading to an understanding at the sequence and structural level as towhat defines a primary or a secondary origin and what environmental or cellular processes participate in the selection ofwhich origin is used. MATERIALS AND METHODSBacterial and Phage Stains. Escherichia coli (sup0) and B40 (supI) were used as hosts for the propagation of T4 phages. T4 phages were from the collection originally maintained at the California Institute of Technology. To obtain cytosine-containing T4 DNA, a multiple mutant ofT4 56-, denA-, denB-alc-(provided by E. Kutter) was propagated on E. coli K803 (supII) and B834 (sup0) (9). The A phage used as a cloning vector was A 569 and it was propagated on E. coli C600 (10).Isolation of Labeled Early T4 DNA. E. coli B was grown at 37'C in minimal medium containing 0.05 M Tris-HC1 (pH 7.4), 0.019 M NH4Cl, 0.017 M NaCl, 3 tkM FeC13, 3 mM MgCl2, 2 mM Na2SO4, 0.5% Casamino acids, 0.5% glucose, and 0.16 mM phosphate. Cells were infected at a density of 5 x 108 ml. At 20 minutes prior to infection, H332P04 (New England Nuclear, 1.5 mCi/ml; 1 Ci = 3.7 x 1010 becquerels) was added to equilibrate the intracellular phosphate pool. The 32P-labeled cells were concentrated 10-fold by centrifuigation and infected withT4D at a multiplicity of1:10. Phage adsorption was allowed for 10 min at 0C. Thereafter, the infected culture was diluted to the original volume with prewarmed medium at 37C. At various times afterward, aliquots were harvested by adding chloramphenicol at 50 ,ug/ml and 10 mM NaN3 and then were quickly frozen in a dry ice/acetone bath. 32P-L...

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Identification of the origins of T4 DNA replication.

King¹,

Huang²

1982

“…Replication ofbacteriophage T4 DNA is initiated on linear DNA molecules at one or several preferred origins (1)(2)(3)(4)(5)(6)(7)(8)(9). Soon after the first initiation, many more replication forks are initiated, leading to a rapid acceleration ofDNA synthesis (10,11).…”

mentioning

confidence: 99%

Two alternative mechanisms for initiation of DNA replication forks in bacteriophage T4: priming by RNA polymerase and by recombination.

Luder¹,

Mosig²

1982

132

We show that bacteriophage T4 has two alternative mechanisms to initiate DNA replication: one dependent on Escherichia coli RNA polymerase (RNA nucleotidyltransferase, EC 2.7.7.6), and one dependent on general recombination. Continued DNA synthesis under recombination-defective conditions was sensitive to rifampin, an inhibitor ofRNA polymerase. On the other hand, DNA synthesis accelerated in spite of the presence of rifampin if recombination occurred.Replication ofbacteriophage T4 DNA is initiated on linear DNA molecules at one or several preferred origins (1-9). Soon after the first initiation, many more replication forks are initiated, leading to a rapid acceleration ofDNA synthesis (10, 11). During this acceleration period, a complex network of DNA is formed (12)(13)(14)(15)(16)(17). This process requires recombination functions (9, 18). Most mutations that inhibit recombination not only prevent for, mation of this network but also arrest DNA synthesis prematurely (19)(20)(21)(22). This indicates that recombination and the continuation ofDNA replication are interdependent (for review see ref. 23). The underlying reasons have remained unknown because of observations seemingly inconsistent with simple explanations: the DNA arrest phenotype ofcertain recombinationdefective mutants is overcome by additional mutations in genes 33 and 55 (20-22, 24), which code for RNA polymerase accessory proteins (25)(26)(27).To explain these and other apparently contradictory results, we have proposed that phage T4 uses different modes to initiate DNA replication: initiation from specific origin sequences, which we define as "primary" initiation, and subsequent "secondary" initiation from recombinational intermediates (7,8,28).For several reasons (7,8,29,30) we suspected that host RNA polymerase (RNA nucleotidyltransferase, EC 2.7.7.6) is required for primary initiation although it has been shown that late wild-type DNA replication does not depend on RNA polymerase (31, 32). After the onset of DNA replication, gene 33 and 55 products associate with RNA polymerase (25-27) to effect the switch to late gene expression (33-35) by altering promoter recognition (36). If unmodified host RNA polymerase were required for primary origin initiation, the association with gene 33 and 55 products would shut offreinitiation from primary origins and make the alternative recombinational initiation indispensable for growth of wild-type T4. This hypothesis accounts for the observations mentioned above: premature arrest of DNA synthesis in recombination-defective mutants (46-47-) and restoration of DNA synthesis by additional mutations in genes 33 and 55. Although the rate of DNA synthesis under these conditions is similar to that ofwild-type T4, no branched concatemers are formed (20)(21)(22). Instead, the DNA replicates as linear molecules of unit length, which we suspected to require continued initiation from primary origins by RNA polymerase. Therefore, this replication should remain sensitive to inhibitors of RNA polymerase at late...

“…The gene 34 hotspot very likely depends on ori (34), because the origin maps near the peak of the hotspot (13) and because ori (34) generates a hotspot when inserted intofrd. Although we have not cloned a tertiary origin that maps within the gene 5 hotspot (14), others have reported the presence of an origin in this region (30,31). Thus, all three major recombination hotspots probably depend on replication origins.…”

Section: Discussionmentioning

confidence: 99%

Recombination hotspots in bacteriophage T4 are dependent on replication origins.

Yap

1991

Bacteriophage T4 recombination "hotspots"were first detected by the rescue of genetic markers from UV-irradiated phage particles. These hotspots have since been detected following treatments that yield other forms of DNA damage, and at least one is active in the absence ofdamage. ing nuclease-induced cleavage of DNA (1). Prokaryotic hotspots are also generated by site-specific recombination systems, including the phage P1 cre/lox and phage A att/int systems (2, 3). The HOT] sequence from the Saccharomyces cerevisiae rRNA gene cluster stimulates mitotic recombination by inducing transcription (4). As judged from these and other examples, hotspot sequences may generally function by either serving as recognition sites for recombination endonucleases or increasing the accessibility of DNA to recombination enzymes. Three major recombination hotspots have been described in the bacteriophage T4 genome (5). Each hotspot will be referred to here using the name of a gene that maps near the hotspot peak (i.e., the gene 5, gene uvsY, and gene 34 hotspots). The hotspots were first detected by marker rescue, a process by which wild-type alleles from inviable UVirradiated donor phage are recovered into viable nonirradiated helper phage carrying amber (am) mutations in the corresponding genes. Recombination within the hotspot regions was -5-fold more frequent than in less recombinogenic areas (Fig. 1). A similar topography of rescue occurs when donor phage are damaged by either x-rays (6) or 32P decay (7), and therefore the hotspots do not arise from a nonrandom distribution of UV-induced damage. In addition, the gene 34 hotspot causes a large increase in recombination between undamaged coinfecting phage genomes (8-11) (see Discussion MATERIALS AND METHODS Materials. Enzymes and EcoRI linkers were purchased from commercial sources. As described previously (18), cells were grown in L broth and EHA plates were used for titering T4 phage. M9S buffer contained KH2PO4 (8 g/liter), Na2HPO4 (6 g/liter), NH4Cl (1 g/liter), and FeCl3 (0.16 mg/liter).Strains. E. coli CR63 (supD) and BE (nonsuppressing, sup0) were the permissive and restrictive hosts for amber mutant phage, respectively. E. coli AB1157 (F-thr-J leuB6 thi-1 lacYl galK2 ara-14 xyl-S mtl-i proA2 kdgKSJ rpsL31 tsx-33 his4 argE3 supE44) and JC10287 [AB1157 with A(srlRrecA)304 mutation] were provided by R. Kolodner (DanaFarber Cancer Institute). T4 strain K10 has the genotype 38a" 51 am denA-A(denB-rII), K10-116 is isogenic except for a linker-insertion mutation in uvs Y, and K10-608 has a deletion that removes the uvs Y promoter (16). The K10 Abbreviation: I/S, insertion substitution.