scholarly journals In vitro reconstitution of DNA replication initiated by genetic recombination: a T4 bacteriophage model for a type of DNA synthesis important for all cells

2019 ◽  
Vol 30 (1) ◽  
pp. 146-159 ◽  
Author(s):  
Jack Barry ◽  
Mei Lie Wong, ◽  
Bruce Alberts
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Genetic Recombination ◽  
Bacteriophage T4 ◽  
Dna Recombination ◽  
Replication Forks ◽  
Dna Molecules ◽  
Bacteriophage Infection ◽  
T4 Bacteriophage ◽  
Dna Double Helix

Using a mixture of 10 purified DNA replication and DNA recombination proteins encoded by the bacteriophage T4 genome, plus two homologous DNA molecules, we have reconstituted the genetic recombination–initiated pathway that initiates DNA replication forks at late times of T4 bacteriophage infection. Inside the cell, this recombination-dependent replication (RDR) is needed to produce the long concatemeric T4 DNA molecules that serve as substrates for packaging the shorter, genome-sized viral DNA into phage heads. The five T4 proteins that catalyze DNA synthesis on the leading strand, plus the proteins required for lagging-strand DNA synthesis, are essential for the reaction, as are a special mediator protein (gp59) and a Rad51/RecA analogue (the T4 UvsX strand-exchange protein). Related forms of RDR are widespread in living organisms—for example, they play critical roles in the homologous recombination events that can restore broken ends of the DNA double helix, restart broken DNA replication forks, and cross over chromatids during meiosis in eukaryotes. Those processes are considerably more complex, and the results presented here should be informative for dissecting their detailed mechanisms.

scholarly journals Involvement of a replicative DNA helicase of bacteriophage T4 in DNA recombination.

Genetics ◽  
1994 ◽  
Vol 138 (2) ◽  
pp. 247-252 ◽  
Author(s):  
T Yonesaki
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Genetic Recombination ◽  
Bacteriophage T4 ◽  
Dna Recombination ◽  
Dna Helicase ◽  
Replicative Helicase ◽  
Helicase Gene ◽  
A Subunit ◽  
Lagging Strand

Abstract Bacteriophage T4 gene 41 encodes a replicative DNA helicase that is a subunit of the primosome which is essential for lagging-strand DNA synthesis. A mutation, rrh, was generated and selected in the helicase gene on the basis of limited DNA replication that ceases early. The survival of ultraviolet-irradiated phage and the frequency of genetic recombination are reduced by rrh. In addition, rrh diminishes the production of concatemeric DNA. These results strongly suggest that the gene 41 replicative helicase participates in DNA recombination.

scholarly journals RecG and UvsW catalyse robust DNA rewinding critical for stalled DNA replication fork rescue

2013 ◽  
Vol 4 (1) ◽  
Author(s):  
Maria Manosas ◽  
Senthil K. Perumal ◽  
Piero R. Bianco ◽  
Felix Ritort ◽  
Stephen J. Benkovic ◽  
...  
Keyword(s):  
Dna Repair ◽  
Dna Replication ◽  
Single Molecule ◽  
Replication Fork ◽  
Genetic Recombination ◽  
Bacteriophage T4 ◽  
General Mechanism ◽  
Dna Unwinding ◽  
Replication Forks ◽  
Displacement Reactions

Abstract Helicases that both unwind and rewind DNA have central roles in DNA repair and genetic recombination. In contrast to unwinding, DNA rewinding by helicases has proved difficult to characterize biochemically because of its thermodynamically downhill nature. Here we use single-molecule assays to mechanically destabilize a DNA molecule and follow, in real time, unwinding and rewinding by two DNA repair helicases, bacteriophage T4 UvsW and Escherichia coli RecG. We find that both enzymes are robust rewinding enzymes, which can work against opposing forces as large as 35 pN, revealing their active character. The generation of work during the rewinding reaction allows them to couple rewinding to DNA unwinding and/or protein displacement reactions central to the rescue of stalled DNA replication forks. The overall results support a general mechanism for monomeric rewinding enzymes.

Single-molecule binding characterization of primosomal protein PriA involved in replication restart

2021 ◽  
Author(s):  
Tzu-Yu Lee ◽  
Yi-Ching Li ◽  
Min-Guan Lin ◽  
Chwan-Deng Hsiao ◽  
Hung-Wen Li
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Single Molecule ◽  
Replication Forks ◽  
Bacterial Survival ◽  
Dna Damages ◽  
Replication Restart

DNA damages lead to stalled or collapsed replication forks. Replication restart primosomes re-initiate DNA synthesis at these stalled or collapsed DNA replication forks, which is important for bacterial survival. Primosomal...

scholarly journals Rad53 controls DNA unwinding after helicase-polymerase uncoupling at DNA replication forks

2019 ◽  
Author(s):  
Sujan Devbhandari ◽  
Dirk Remus
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Point Mutations ◽  
Dna Helicase ◽  
Dna Unwinding ◽  
Replication Forks ◽  
Chromosomal Dna ◽  
Replication Checkpoint ◽  
Polymerase Domain ◽  
Tightly Coupled

ABSTRACTThe coordination of DNA unwinding and synthesis at replication forks promotes efficient and faithful replication of chromosomal DNA. Using the reconstituted budding yeast DNA replication system, we demonstrate that Pol ε variants harboring catalytic point mutations in the Pol2 polymerase domain, contrary to Pol2 polymerase domain deletions, inhibit DNA synthesis at replication forks by displacing Pol δ from PCNA/primer-template junctions, causing excessive DNA unwinding by the replicative DNA helicase, CMG, uncoupled from DNA synthesis. Mutations that suppress the inhibition of Pol δ by Pol ε restore viability in Pol2 polymerase point mutant cells. We also observe uninterrupted DNA unwinding at replication forks upon dNTP depletion or chemical inhibition of DNA polymerases, demonstrating that leading strand synthesis is not tightly coupled to DNA unwinding by CMG. Importantly, the Rad53 kinase controls excessive DNA unwinding at replication forks by limiting CMG helicase activity, suggesting a mechanism for fork-stabilization by the replication checkpoint.

scholarly journals Two New Early Bacteriophage T4 Genes,repEA and repEB, That Are Important for DNA Replication Initiated from Origin E

1999 ◽  
Vol 181 (22) ◽  
pp. 7115-7125 ◽  
Author(s):  
Rita Vaiskunaite ◽  
Andrew Miller ◽  
Laura Davenport ◽  
Gisela Mosig
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Bacteriophage T4 ◽  
Direct Synthesis ◽  
Growth Conditions ◽  
Dna Strands ◽  
Different Temperatures ◽  
And Function ◽  
Different Origins

ABSTRACT Two new, small, early bacteriophage T4 genes, repEA andrepEB, located within the origin E (oriE) region of T4 DNA replication, affect functioning of this origin. An important and unusual property of the oriE region is that it is transcribed at early and late periods after infection, but in opposite directions (from complementary DNA strands). The early transcripts are mRNAs for RepEA and RepEB proteins, and they can serve as primers for leading-strand DNA synthesis. The late transcripts, which are genuine antisense RNAs for the early transcripts, direct synthesis of virion components. Because the T4 genome contains several origins, and because recombination can bypass a primase requirement for retrograde synthesis, neither defects in a single origin nor primase deficiencies are lethal in T4 (Mosig et al., FEMS Microbiol. Rev. 17:83–98, 1995). Therefore, repEA and repEBwere expected and found to be important for T4 DNA replication only when activities of other origins were reduced. To investigate the in vivo roles of the two repE genes, we constructed nonsense mutations in each of them and combined them with the motAmutation sip1 that greatly reduces initiation from other origins. As expected, T4 DNA synthesis and progeny production were severely reduced in the double mutants as compared with the singlemotA mutant, but early transcription of oriEwas reduced neither in the motA nor in the repEmutants. Moreover, residual DNA replication and growth of the double mutants were different at different temperatures, suggesting different functions for repEA and repEB. We surmise that the different structures and protein requirements for functioning of the different origins enhance the flexibility of T4 to adapt to varied growth conditions, and we expect that different origins in other organisms with multiorigin chromosomes might differ in structure and function for similar reasons.

scholarly journals EXPRESSION OF A DNA REPLICATION GENE CLUSTER IN BACTERIOPHAGE T4: GENETIC LINKAGE AND THE CONTROL OF GENE PRODUCT INTERACTIONS

Genetics ◽  
1984 ◽  
Vol 107 (4) ◽  
pp. 537-549 ◽  
Author(s):  
W L Gerald ◽  
J D Karam
Keyword(s):  
Dna Replication ◽  
Genetic Linkage ◽  
Bacteriophage T4 ◽  
Inhibitory Effect ◽  
Wild Type ◽  
Essential Components ◽  
T4 Bacteriophage ◽  
Missense Mutant ◽  
Type Gene ◽  
And Control

ABSTRACT The results of this study bear on the relationship between genetic linkage and control of interactions between the protein products of different cistrons. In T4 bacteriophage, genes 45 and 44 encode essential components of the phage DNA replication multiprotein complex. T4 gene 45 maps directly upstream of gene 44 relative to the overall direction of reading of this region of the phage chromosome, but it is not known whether these two genes are cotranscribed. It has been shown that a nonsense lesion of T4 gene 45 exerts a cis-dominant inhibitory effect on growth of a missense mutant of gene 44 but not on growth of phage carrying the wild-type gene 44 allele. In previous work, we confirmed these observations on polarity of the gene 45 mutation but detected no polar effects by this lesion on synthesis of either mutant or wild-type gene 44 protein. In the present study, we demonstrate that mRNA for gene 44 protein is separable by gel electrophoresis from gene 45-protein-encoding mRNA. That is, the two proteins are not synthesized from one polycistronic message, and the cis-dominant inhibitory effect of the gene 45 mutation on gene 44 function is probably expressed at a posttranslational stage. We propose that close genetic linkage, whether or not it provides shared transcriptional and translational regulatory signals for certain clusters of functionally related cistrons, may determine the intracellular compartmentalization for synthesis of proteins encoded by these clusters. In prokaryotes, such linkage-dependent compartmentation may minimize the diffusion distances between gene products that are synthesized at low levels and are destined to interact.

scholarly journals The integrated stress response induces R-loops and hinders replication fork progression

2020 ◽  
Author(s):  
Josephine Ann Mun Yee Choo ◽  
Denise Schlösser ◽  
Valentina Manzini ◽  
Anna Magerhans ◽  
Matthias Dobbelstein
Keyword(s):  
Protein Synthesis ◽  
Dna Replication ◽  
Stress Response ◽  
Dna Synthesis ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Replication Forks ◽  
Integrated Stress Response ◽  
Loop Formation ◽  
Initiation Of Translation

ABSTRACTThe integrated stress response (ISR) allows cells to rapidly shut down most of their protein synthesis in response to protein misfolding, amino acid deficiency, or virus infection. These stresses trigger the phosphorylation of the translation initiation factor eIF2alpha, which prevents the initiation of translation. Here we show that triggering the ISR drastically reduces the progression of DNA replication forks within one hour, thus flanking the shutdown of protein synthesis with immediate inhibition of DNA synthesis. DNA replication is restored by compounds that inhibit eIF2alpha kinases or re-activate eIF2alpha. Mechanistically, the translational shutdown blocks histone synthesis, promoting the formation of DNA:RNA hybrids (R-loops) which interfere with DNA replication. Histone depletion alone induces R-loops and compromises DNA replication. Conversely, histone overexpression or R-loop removal by RNaseH1 each restores DNA replication in the context of ISR and histone depletion. In conclusion, the ISR rapidly stalls DNA synthesis through histone deficiency and R-loop formation. We propose that this shutdown mechanism prevents potentially detrimental DNA replication in the face of cellular stresses.SIGNIFICANCEThe integrated stress response has long been explored regarding its immediate impact on protein synthesis. Translational shutdown represents an indispensable mechanism to prevent the toxicity of misfolded proteins and virus infections. Our results indicate that the shutdown mechanisms reach far beyond translation and immediately interfere with DNA synthesis as well. ISR depletes cells of new histones which induce accumulation of DNA:RNA hybrids. The impairment of DNA replication in this context supports cell survival during stress.Our work provides a link between the ISR and another subject of active research, i. e. the regulatory network of DNA replication forks.Graphical Abstract

scholarly journals Phosphorylation of PNKP mediated by CDKs promotes end-processing of Okazaki fragments during DNA replication

2021 ◽  
Author(s):  
Kaima Tsukada ◽  
Rikiya Imamura ◽  
Kotaro Saikawa ◽  
Mizuki Saito ◽  
Naoya Kase ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Dna Replication ◽  
Dna Synthesis ◽  
High Speed ◽  
Replication Forks ◽  
Cyclin Dependent Kinases ◽  
Okazaki Fragments ◽  
Dna Ligation ◽  
Polynucleotide Kinase ◽  
Dna Ends

Polynucleotide kinase phosphatase (PNKP) has enzymatic activities as 3′ phosphatase and 5′ kinase of DNA ends to promote DNA ligation. Here, we show that PNKP is involved in progression of DNA replication through end-processing of Okazaki fragments (OFs). Cyclin-dependent kinases (CDKs) regulate phosphorylation on threonine 118 (T118) of PNKP, and which phosphorylation allows it to be recruited to OFs. Loss of PNKP and T118 phosphorylation significantly increased unligated OFs and high-speed DNA synthesis in replication forks, suggesting that PNKP T118 phosphorylation is required for OFs ligation for its maturation. Furthermore, phosphatase-dead PNKP also exhibited an accumulation of unligated OFs and high-speed DNA synthesis. Overall, our data suggested that CDK-mediated PNKP phosphorylation at T118 is important for its recruitment to OFs and PNKP subsequently promotes end-processing for OFs maturation for stable cell proliferation.

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