Endonuclease VII resolves Y-junctions in branched DNA in vitro.

Jensch, F.; Kemper, Börries

doi:10.1002/j.1460-2075.1986.tb04194.x

Cited by 102 publications

(55 citation statements)

References 33 publications

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“…These include, among others, three-way junctions (Y structures) such as occur at the replication fork during DNA replication. Endo VII has been shown to resolve a variety of synthetically constructed Y structures into linear molecules by introducing nicks 3Ј to the junction (15,33). To our knowledge, however, endo VII cleavage of naturally occurring RIs has not been investigated so far.…”

Section: Resultsmentioning

confidence: 99%

Architecture of the Replication Fork Stalled at the 3′ End of Yeast Ribosomal Genes

Gruber¹,

Wellinger²,

Sogo³

2000

Molecular and Cellular Biology

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Every unit of the rRNA gene cluster of Saccharomyces cerevisiae contains a unique site, termed the replication fork barrier (RFB), where progressing replication forks are stalled in a polar manner. In this work, we determined the positions of the nascent strands at the RFB at nucleotide resolution. Within an HpaI-HindIII fragment essential for the RFB, a major and two closely spaced minor arrest sites were found. In the majority of molecules, the stalled lagging strand was completely processed and the discontinuously synthesized nascent lagging strand was extended three bases farther than the continuously synthesized leading strand. A model explaining these findings is presented. Our analysis included for the first time the use of T4 endonuclease VII, an enzyme recognizing branched DNA molecules. This enzyme cleaved predominantly in the newly synthesized homologous arms, thereby specifically releasing the leading arm.DNA replication is a complex process that can be divided into three steps: initiation, ongoing replication, and termination. Whereas extensive work has been done on the first of these steps, only little is known about the last, even though hundreds of termination events occur every time the genome of an eukaryotic organism is replicated. Replication termination in the chromosomes of Saccharomyces cerevisiae seems to occur throughout a broad region, whenever two converging replication forks meet, rather than at specific sites (4, 30). An exception to this rule is represented by the specific replication termination site at the 3Ј end of the rRNA transcription unit, the replication fork barrier (RFB). The RFB was originally found in the yeast S. cerevisiae (5, 22) and subsequently observed in many disparate organisms including Schizosaccharomyces pombe, frog, mouse, human, and plant (12,13,23,24,36,45). Thus, the RFB seems to be a common feature of eukaryotic rDNA replication (13). The location of the stalled fork at the RFB in S. cerevisiae was narrowed down to a 129-bp restriction fragment defined by a HindIII-HpaI restriction site ( Fig. 1; see also reference 6). Furthermore, the sequences required for the block seem to reside within this fragment, even though additional sequences could be necessary to restore the full functionality of the RFB (6, 19).The molecular mechanism of the RFB, however, remains elusive. It has been demonstrated that transcription elongation is not a prerequisite for a functional RFB, since the blockage was observed in a strain lacking RNA polymerase I (6). However, in this yeast strain even nucleosome-free enhancers have been detected which can have RFB activity (9). Moreover, sequences close to the 3Ј end of the 35S rRNA gene (rDNA) retained their blocking ability when inserted into extrachromosomal plasmids lacking the transcription unit (6, 19). On the other hand, these sequences did not impede replication fork movement on a plasmid in Escherichia coli, excluding possible structures inherent in the DNA sequence. This finding led to the suggestion that a specific protein ...

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Section: Resultsmentioning

confidence: 99%

Architecture of the Replication Fork Stalled at the 3′ End of Yeast Ribosomal Genes

Gruber¹,

Wellinger²,

Sogo³

2000

Molecular and Cellular Biology

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“…This decrease is consistent with EndoVII-mediated cleavage of the regressed fork Holliday junction. However, previous reports have suggested that EndoVII also cleaves Y-shaped molecules, such as the origin fork, in vivo (14,15). Direct cleavage of the origin fork junction by EndoVII would decrease the number of origin forks available for regression, indirectly decreasing the accumulation of regressed origin forks.…”

Section: Accumulation and Resolution Of Origin Forks In Various Mutantmentioning

confidence: 95%

“…The major function of EndoVII appears to be the resolution of branched molecules before packaging of DNA into phage heads. In contrast to some other resolvases, such as RuvC, EndoVII can act on a broad range of DNA structures in vitro, including fork-shaped structures (14)(15)(16).…”

mentioning

confidence: 99%

Regression supports two mechanisms of fork processing in phage T4

Long

Kreuzer

2008

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

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Replication forks routinely encounter damaged DNA and tightly bound proteins, leading to fork stalling and inactivation. To complete DNA synthesis, it is necessary to remove fork-blocking lesions and reactivate stalled fork structures, which can occur by multiple mechanisms. To study the mechanisms of stalled fork reactivation, we used a model fork intermediate, the origin fork, which is formed during replication from the bacteriophage T4 origin, ori(34). The origin fork accumulates within the T4 chromosome in a site-specific manner without the need for replication inhibitors or DNA damage. We report here that the origin fork is processed in vivo to generate a regressed fork structure. Furthermore, origin fork regression supports two mechanisms of fork resolution that can potentially lead to fork reactivation. Fork regression generates both a site-specific double-stranded end (DSE) and a Holliday junction. Each of these DNA elements serves as a target for processing by the T4 ATPase/exonuclease complex [gene product (gp) 46/47] and Holliday junction-cleaving enzyme (EndoVII), respectively. In the absence of both gp46 and EndoVII, regressed origin forks are stabilized and persist throughout infection. In the presence of EndoVII, but not gp46, there is significantly less regressed origin fork accumulation apparently due to cleavage of the regressed fork Holliday junction. In the presence of gp46, but not EndoVII, regressed origin fork DSEs are processed by degradation of the DSE and a pathway that includes recombination proteins. Although both mechanisms can occur independently, they may normally function together as a single fork reactivation pathway.2D gel electrophoresis ͉ DNA replication ͉ recombination ͉ restart R eplication forks routinely encounter various lesions, ranging from damaged nucleotide residues to covalent protein-DNA complexes. The consequences of these encounters depend on the type and position of the lesion (1). Although some lesions result in fork breakage, many result in fork stalling and replisome inactivation. Presumably, replisome disassembly is important for the repair of the original DNA lesion. However, it is then necessary to reactivate stalled forks to complete replication and maintain genome stability. Therefore, it is critical to understand the mechanisms of stalled fork resolution and reactivation.To identify the pathways of stalled fork reactivation, we have used a model fork intermediate that is formed during replication from bacteriophage T4 origin, ori(34). Replication from ori(34) occurs bidirectionally through a two-step process (Fig. 1) (2). A middle-mode promoter and DNA unwinding element promote the formation of an R loop that initiates the first replication fork (3). The mechanism of T4 replication from an R loop has been analyzed in vitro (4). Initially, the branch structure of the R loop supports the binding of the replicative helicase loader [gene product (gp) 59], which promotes removal of the ssDNA-binding protein (gp32) in favor of the replicative helicase (gp41). ...

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“…This enzyme also cleaves synthetic Y structures composed of three complementary oligonucleotides (46,47), suggesting that it could play a role in the processing of blocked replication forks in vivo. To begin to test this hypothesis, we compared blocked forks induced by m-AMSA in T4 wild-type versus T4 gene 49 amber-mutant (T4 49 am ) infections of a nonsuppressing host.…”

Section: Accumulation Of Blocked Replication Forks In Gene 49mentioning

confidence: 99%

Endonuclease cleavage of blocked replication forks: An indirect pathway of DNA damage from antitumor drug–topoisomerase complexes

Hong

Kreuzer

2003

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

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The cytotoxicity of several important antitumor drugs depends on formation of the covalent topoisomerase-DNA cleavage complex. However, cellular processes such as DNA replication are necessary to convert the cleavage complex into a cytotoxic lesion, but the molecular mechanism of this conversion and the precise nature of the cytotoxic lesion are unknown. Using a bacteriophage T4 model system, we have previously shown that antitumor drug-induced cleavage complexes block replication forks in vivo. In this report, we show that these blocked forks can be cleaved by T4 endonuclease VII to create overt DNA breaks. The accumulation of blocked forks increased in endonuclease VII-deficient infections, suggesting that endonuclease cleavage contributes to fork processing in vivo. Furthermore, purified endonuclease VII cleaved the blocked forks in vitro close to the branch points. These results suggest that an indirect pathway of branched-DNA cleavage contributes to the cytotoxicity of antitumor drugs that target DNA topoisomerases. S everal important classes of antitumor drugs, as well as the antibacterial quinolones, inhibit type II topoisomerases by a common mechanism of action (1-3). These drugs alter the equilibrium of the reaction cycle to favor an otherwise transient reaction intermediate called the cleavage complex. Within the cleavage complex, the enzyme is covalently linked, by means of phosphotyrosine bonds, to the cleaved DNA at two staggered positions on the opposing strands of the helix. Camptothecin and related anticancer drugs attack eukaryotic topoisomerase I by a related mechanism, trapping a cleavage complex with a single cleaved phosphodiester bond (4).Various results demonstrate that cytotoxicity of type II topoisomerase inhibitors depends on formation of the cleavage complex, and not just inhibition of enzyme activity (2, 5, 6). However, the cleavage complex itself is reversible, and is thus unlikely to be the cytotoxic lesion (7). Instead, cellular processes apparently convert the cleavage complex into a cytotoxic lesion (8,9). DNA replication appears to play an important role in this conversion because S phase cells are more sensitive to topoisomerase inhibitors than G 1 cells, and because the replication inhibitor aphidicolin can abrogate drug sensitivity (8-11). Despite the implication that DNA replication is involved in cytotoxicity, the exact nature of the cytotoxic lesion and the pathway of its formation are not clear.A drug-induced cleavage complex with a type II topoisomerase contains a latent double-strand DNA break (DSB), but in vitro this break is revealed only when the protein is denatured with a strong detergent such as SDS (see ref. 5). Nonetheless, overt DNA breaks are apparently generated from the druginduced cleavage complexes in vivo, and these breaks are probably important in cytotoxicity. Several types of indirect evidence support this view. First, mutants deficient in DSB repair are hypersensitive to topoisomerase inhibitors that induce the cleavage complex (12-15). Second, cleava...

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Endonuclease VII resolves Y-junctions in branched DNA in vitro.

Cited by 102 publications

References 33 publications

Architecture of the Replication Fork Stalled at the 3′ End of Yeast Ribosomal Genes

Architecture of the Replication Fork Stalled at the 3′ End of Yeast Ribosomal Genes

Regression supports two mechanisms of fork processing in phage T4

Endonuclease cleavage of blocked replication forks: An indirect pathway of DNA damage from antitumor drug–topoisomerase complexes

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