scholarly journals A new role for Holliday junction resolvase Yen1 in processing DNA replication intermediates exposes Dna2 as an accessory replicative helicase

2017 ◽  
Vol 4 (1) ◽  
pp. 32-34 ◽  
Author(s):  
Benoit Falquet ◽  
Ulrich Rass
Keyword(s):  
Dna Replication ◽  
Holliday Junction ◽  
Replicative Helicase ◽  
Holliday Junction Resolvase ◽  
Replication Intermediates

scholarly journals Repression of Vaccinia Virus Holliday Junction Resolvase Inhibits Processing of Viral DNA into Unit-Length Genomes

2001 ◽  
Vol 75 (14) ◽  
pp. 6460-6471 ◽  
Author(s):  
Alonzo D. Garcia ◽  
Bernard Moss
Keyword(s):  
Dna Replication ◽  
Vaccinia Virus ◽  
Gel Electrophoresis ◽  
Unit Length ◽  
Polyacrylamide Gel Electrophoresis ◽  
Holliday Junction ◽  
Application Site ◽  
Viral Dna ◽  
Viral Protein Synthesis ◽  
Holliday Junction Resolvase

ABSTRACT The vaccinia virus A22R gene encodes a protein that is homologous to the bacterial enzyme RuvC and specifically cleaves and resolves four-way DNA Holliday junctions into linear duplex products. To investigate the role of the vaccinia virus Holliday junction resolvase during an infection, we constructed two recombinant viruses: vA22-HA, which has a short C-terminal epitope tag appended to the A22R open reading frame, and vA22i, in which the original A22R gene is deleted and replaced by an inducible copy. Polyacrylamide gel electrophoresis and Western blot analysis of extracts and purified virions from cells infected with vA22-HA revealed that the resolvase was expressed after the onset of DNA replication and incorporated into virion cores. vA22i exhibited a conditional replication defect. In the absence of an inducer, (i) viral protein synthesis was unaffected, (ii) late-stage viral DNA replication was reduced, (iii) most of the newly synthesized viral DNA remained in a branched or concatemeric form that caused it to be trapped at the application site during pulsed-field gel electrophoresis, (iv) cleavage of concatemer junctions was inhibited, and (v) virion morphogenesis was arrested at an immature stage. These data indicated multiple roles for the vaccinia virus Holliday junction resolvase in the replication and processing of viral DNA into unit-length genomes.

scholarly journals Canonical and novel non-canonical activities of the Holliday junction resolvase Yen1

2020 ◽  
Author(s):  
F. Javier Aguado ◽  
Raquel Carreira ◽  
Vanesa Hurtado-Nieves ◽  
Miguel G. Blanco
Keyword(s):  
Budding Yeast ◽  
Holliday Junction ◽  
Conformational Preference ◽  
Alternative Processing ◽  
Holliday Junction Resolvase ◽  
Replication Intermediates

ABSTRACTYen1 and GEN1 are members of the Rad2/XPG family of nucleases that were identified as the first canonical nuclear Holliday junction (HJ) resolvases in budding yeast and humans due to their ability to introduce two symmetric, coordinated incisions on opposite strands of the HJ, yielding nicked DNA products that could be readily ligated. While GEN1 has been extensively characterized in vitro, much less is known about the biochemistry of Yen1. Here, we have performed the first in-depth characterization of purified Yen1. We confirmed that Yen1 resembles GEN1 in many aspects, including range of substrates targeted, position of most incisions they produce or monomeric state in solution. However, we have also observed unexpected alternative processing of substrates, such as nicked HJs and a different conformational preference on intact HJs. Moreover, we demonstrate that Yen1 is endowed with additional nuclease activities, like a nick-specific 5’-3’ exonuclease or HJ arm-chopping that could apparently blur its classification as a canonical HJ resolvase. Despite this, we show that Yen1 fulfills the requirements of a canonical HJ resolvase and hypothesize that its wider array of nuclease activities might contribute to its function in the removal of persistent recombination or replication intermediates.

scholarly journals Replication intermediates that escape Dna2 activity are processed by Holliday junction resolvase Yen1

2016 ◽  
Vol 7 (1) ◽  
Author(s):  
Gizem Ölmezer ◽  
Maryna Levikova ◽  
Dominique Klein ◽  
Benoît Falquet ◽  
Gabriele Alessandro Fontana ◽  
...  
Keyword(s):  
Holliday Junction ◽  
Holliday Junction Resolvase ◽  
Replication Intermediates

Altered formation of DNA replication intermediates in cells growing in different culture conditions

1989 ◽  
Vol 138 (1) ◽  
pp. 45-49
Author(s):  
Ulf lönn ◽  
Sigrid Lönn ◽  
Urban Nylen ◽  
Gerard Winblad
Keyword(s):  
Dna Replication ◽  
Culture Conditions ◽  
Replication Intermediates ◽  
In Cells

scholarly journals Changes in the topology of DNA replication intermediates: Important discrepancies between in vitro and in vivo

BioEssays ◽  
2021 ◽  
Vol 43 (5) ◽  
pp. 2000309
Author(s):  
Jorge B. Schvartzman ◽  
Víctor Martínez ◽  
Pablo Hernández ◽  
Dora B. Krimer ◽  
María‐José Fernández‐Nestosa
Keyword(s):  
Dna Replication ◽  
Replication Intermediates

scholarly journals UV-induced Hyperphosphorylation of Replication Protein A Depends on DNA Replication and Expression of ATM Protein

2001 ◽  
Vol 12 (5) ◽  
pp. 1199-1213 ◽  
Author(s):  
Gregory G. Oakley ◽  
Lisa I. Loberg ◽  
Jiaqin Yao ◽  
Mary A. Risinger ◽  
Remy L. Yunker ◽  
...  
Keyword(s):  
Dna Replication ◽  
Uv Radiation ◽  
Excision Repair ◽  
Genetic Disorder ◽  
Protein A ◽  
Dna Recombination ◽  
Delayed Response ◽  
Replication Intermediates

Exposure to DNA-damaging agents triggers signal transduction pathways that are thought to play a role in maintenance of genomic stability. A key protein in the cellular processes of nucleotide excision repair, DNA recombination, and DNA double-strand break repair is the single-stranded DNA binding protein, RPA. We showed previously that the p34 subunit of RPA becomes hyperphosphorylated as a delayed response (4–8 h) to UV radiation (10–30 J/m2). Here we show that UV-induced RPA-p34 hyperphosphorylation depends on expression of ATM, the product of the gene mutated in the human genetic disorder ataxia telangiectasia (A-T). UV-induced RPA-p34 hyperphosphorylation was not observed in A-T cells, but this response was restored by ATM expression. Furthermore, purified ATM kinase phosphorylates the p34 subunit of RPA complex in vitro at many of the same sites that are phosphorylated in vivo after UV radiation. Induction of this DNA damage response was also dependent on DNA replication; inhibition of DNA replication by aphidicolin prevented induction of RPA-p34 hyperphosphorylation by UV radiation. We postulate that this pathway is triggered by the accumulation of aberrant DNA replication intermediates, resulting from DNA replication fork blockage by UV photoproducts. Further, we suggest that RPA-p34 is hyperphosphorylated as a participant in the recombinational postreplication repair of these replication products. Successful resolution of these replication intermediates reduces the accumulation of chromosomal aberrations that would otherwise occur as a consequence of UV radiation.

scholarly journals DNA Unwinding Is an MCM Complex-dependent and ATP Hydrolysis-dependent Process

2004 ◽  
Vol 279 (44) ◽  
pp. 45586-45593 ◽  
Author(s):  
David Shechter ◽  
Carol Y. Ying ◽  
Jean Gautier
Keyword(s):  
Dna Replication ◽  
Neutralizing Antibodies ◽  
Atp Hydrolysis ◽  
Initial Period ◽  
Dna Helicase ◽  
Dna Unwinding ◽  
Origin Firing ◽  
Replicative Helicase ◽  
Mcm Proteins

Minichromosome maintenance proteins (Mcm) are essential in all eukaryotes and are absolutely required for initiation of DNA replication. The eukaryotic and archaeal Mcm proteins have conserved helicase motifs and exhibit DNA helicase and ATP hydrolysis activitiesin vitro. Although the Mcm proteins have been proposed to be the replicative helicase, the enzyme that melts the DNA helix at the replication fork, their function during cellular DNA replication elongation is still unclear. Using nucleoplasmic extract (NPE) fromXenopus laeviseggs and six purified polyclonal antibodies generated against each of theXenopusMcm proteins, we have demonstrated that Mcm proteins are required during DNA replication and DNA unwinding after initiation of replication. Quantitative depletion of Mcms from the NPE results in normal replication and unwinding, confirming that Mcms are required before pre-replicative complex assembly and dispensable thereafter. Replication and unwinding are inhibited when pooled neutralizing antibodies against the six different Mcm2–7 proteins are added during NPE incubation. Furthermore, replication is blocked by the addition of the Mcm antibodies after an initial period of replication in the NPE, visualized by a pulse of radiolabeled nucleotide at the same time as antibody addition. Addition of the cyclin-dependent kinase 2 inhibitor p21cip1specifically blocks origin firing but does not prevent helicase action. When p21cip1is added, followed by the non-hydrolyzable analog ATPγS to block helicase function, unwinding is inhibited, demonstrating that plasmid unwinding is specifically attributable to an ATP hydrolysis-dependent function. These data support the hypothesis that the Mcm protein complex functions as the replicative helicase.

A Test of the Double-Strand Break Repair Model for Meiotic Recombination in Saccharomyces cerevisiae

Genetics ◽  
1996 ◽  
Vol 144 (1) ◽  
pp. 27-41 ◽  
Author(s):  
Larry A Gilbertson ◽  
Franklin W Stahl
Keyword(s):  
Meiotic Recombination ◽  
Strand Break ◽  
Double Strand Break ◽  
Double Strand Break Repair ◽  
Holliday Junction ◽  
Single Event ◽  
Heteroduplex Dna ◽  
Holliday Junction Resolvase ◽  
Break Repair ◽  
Two Sides

Abstract We tested predictions of the double-strand break repair (DSBR) model for meiotic recombination by examining the segregation patterns of small palindromic insertions, which frequently escape mismatch repair when in heteroduplex DNA. The palindromes flanked a well characterized DSB site at the ARC4 locus. The “canonical” DSBR model, in which only 5′ ends are degraded and resolution of the four-stranded intermediate is by Holliday junction resolvase, predicts that hDNA will frequently occur on both participating chromatids in a single event. Tetrads reflecting this configuration of hDNA were rare. In addition, a class of tetrads not predicted by the canonical DSBR model was identified. This class represented events that produced hDNA in a “trans” configuration, on opposite strands of the same duplex on the two sides of the DSB site. Whereas most classes of convertant tetrads had typical frequencies of associated crossovers, tetrads with trans hDNA were parental for flanking markers. Modified versions of the DSBR model, including one that uses a topoisomerase to resolve the canonical DSBR intermediate, are supported by these data.

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