Genetic Analysis of Dna Replication in Bacteriophage T4

Mosig, Gisela; Benedict, Stephen H.; Ghosal, Debabrota; Luder, Andreas; Dannenberg, Richard; Bock, Susan

doi:10.1016/b978-0-12-048850-6.50050-8

Cited by 10 publications

(12 citation statements)

References 34 publications

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“…3, compare lanes A and B). These results are expected as a consequence of the deficiencies in gp4l and gp46: leading-strand initiation at primary origins occurs, but lagging-strand synthesis and recombination-dependent initiation are considerably delayed (19,45 A UC-GG rho' cells (Fig. 3, lanes C and D), but there was less total T4 DNA synthesis in rhoO26 than in rho' cells.…”

Section: H]thymidine Incorporation Experimentsmentioning

confidence: 71%

Impaired expression of certain prereplicative bacteriophage T4 genes explains impaired T4 DNA synthesis in Escherichia coli rho (nusD) mutants

Stitt

Mosig

1989

J Bacteriol

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The Escherichia coli rhoO26 mutation that alters the transcription termination protein Rho prevents growth of wild-type bacteriophage T4. Among the consequences of this mutation are delayed and reduced T4 DNA replication. We show that these defects can be explained by defective synthesis of certain T4 replicationrecombination proteins. Expression of T4 gene 41 (DNA helicase/primase) is drastically reduced, and expression of T4 genes 43 (DNA polymerase), 30 (DNA ligase), 46 (recombination nuclease), and probably 44 (DNA polymerase-associated ATPase) is reduced to a lesser extent. The compensating T4 mutation goFI partially restores the synthesis of these proteins and, concomitantly, the synthesis of T4 DNA in the E. coli rho mutant. From analyzing DNA synthesis in wild-type and various multiply mutant T4 strains, we infer that defective or reduced synthesis of these proteins in rhoO26-infected cells has several major effects on DNA replication. It impairs lagging-strand synthesis during the primary mode of DNA replication; it delays and depresses recombination-dependent (secondary mode) initiation; and it inhibits the use of tertiary origins. All three T4 genes whose expression is reduced in rhoO26 cells and whose upstream sequences are known have a palindrome containing a CUUCGG sequence between the promoter(s) and ribsome-binding site. We speculate that these palindromes might be important for factor-dependent transcription termination-antitermination during normal T4 development. Our results are consistent with previous proposals that the altered Rho factor of rhoO26 may cause excessive termination because the transcription complex does not interact normally with a T4 antiterminator encoded by the wild-type goF gene and that the T4 goFI mutation restores this interaction.Transcription termination factor Rho plays an important role in regulating gene expression in Escherichia coli and its bacteriophages (for reviews, see references 7, 13, 52, and 69). Some E. coli mutants with defective Rho factors show reduced transcription termination at Rho-dependent termination sites and thus suppress transcriptional polar effects. These mutants can also partly overcome the effects of defective lambda gene N antiterminator function at the lambda Rho-dependent termination sites. In contrast, another class of rho mutants, sometimes called nusD, has a different phenotype.' These mutants do not support the normal growth of wild-type bacteriophage lambda or T4, but their defects can be partially suppressed by mutations in phage antiterminator genes: N in lambda and probably goF, comC-ot, or go-9H (which are probably the same genes [23,64,66]) in T4. It is thought that in these rho mutants, the altered protein is unable to respond to phage-encoded antitermination functions (10, 55, 64).The T4 goF gene is dispensable in many hosts; it maps in a nonessential part of the T4 genome (64, 66). Presumably, in wild-type hosts T4 is far less dependent than phage lambda on antitermination functions because many of the T4 delayed-early gene...

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Section: H]thymidine Incorporation Experimentsmentioning

confidence: 71%

Impaired expression of certain prereplicative bacteriophage T4 genes explains impaired T4 DNA synthesis in Escherichia coli rho (nusD) mutants

Stitt

Mosig

1989

J Bacteriol

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“…In 1980, Mosig hypothesized that the late T4 replication is primed by homologous recombination (18), the idea being later confirmed in several ways (19)(20)(21). Kenneth Kreuzer described a particular T4-driven system in which the replication substrate is a circular plasmid that stops origin-dependent replication during the T4 infection, but resumes replication if T4 and the plasmid share homology (20,22).…”

Section: Homologous Recombination In Phage T4mentioning

confidence: 89%

DNA replication meets genetic exchange: Chromosomal damage and its repair by homologous recombination

Kuzminov

2001

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

198

126

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Proceedings of the National Academy of Sciences Colloquium on the roles of homologous recombination in DNA replication are summarized. Current findings in experimental systems ranging from bacteriophages to mammalian cell lines substantiate the idea that homologous recombination is a system supporting DNA replication when either the template DNA is damaged or the replication machinery malfunctions. There are several lines of supporting evidence: (i) DNA replication aggravates preexisting DNA damage, which then blocks subsequent replication; (ii) replication forks abandoned by malfunctioning replisomes become prone to breakage; (iii) mutants with malfunctioning replisomes or with elevated levels of DNA damage depend on homologous recombination; and (iv) homologous recombination primes DNA replication in vivo and can restore replication fork structures in vitro. The mechanisms of recombinational repair in bacteriophage T4, Escherichia coli, and Saccharomyces cerevisiae are compared. In vitro properties of the eukaryotic recombinases suggest a bigger role for single-strand annealing in the eukaryotic recombinational repair.

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“…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).…”

mentioning

confidence: 99%

“…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).…”

mentioning

confidence: 99%

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

Luder¹,

Mosig²

1982

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

132

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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...

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Genetic Analysis of Dna Replication in Bacteriophage T4

Cited by 10 publications

References 34 publications

Impaired expression of certain prereplicative bacteriophage T4 genes explains impaired T4 DNA synthesis in Escherichia coli rho (nusD) mutants

Impaired expression of certain prereplicative bacteriophage T4 genes explains impaired T4 DNA synthesis in Escherichia coli rho (nusD) mutants

DNA replication meets genetic exchange: Chromosomal damage and its repair by homologous recombination

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

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