scholarly journals Effects of DNA Structure and Homology Length on Vaccinia Virus Recombination

2001 ◽  
Vol 75 (15) ◽  
pp. 6923-6932 ◽  
Author(s):  
Xiao-Dan Yao ◽  
David H. Evans
Keyword(s):  
Vaccinia Virus ◽  
Dna Sequences ◽  
Accurate Method ◽  
Single Strand ◽  
Recombination Mechanism ◽  
Virus Recombination ◽  
Single Strand Annealing ◽  
Strand Annealing

ABSTRACT Replicating poxviruses catalyze high-frequency recombination reactions by a process that is not well understood. Using transfected DNA substrates we show that these viruses probably use a single-strand annealing recombination mechanism. Plasmids carrying overlapping portions of a luciferase gene expression cassette and luciferase assays were first shown to provide an accurate method of assaying recombinant frequencies. We then transfected pairs of DNAs into virus-infected cells and monitored the efficiencies of linear-by-linear, linear-by-circle, and circle-by-circle recombination. These experiments showed that vaccinia virus recombination systems preferentially catalyze linear-by-linear reactions much more efficiently than circle-by-circle reactions and catalyze circle-by-circle reactions more efficiently than linear-by-circle reactions. Reactions involving linear substrates required surprisingly little sequence identity, with only 16-bp overlaps still permitting ∼4% recombinant production. Masking the homologies by adding unrelated DNA sequences to the ends of linear substrates inhibited recombination in a manner dependent upon the number of added sequences. Circular molecules were also recombined by replicating viruses but at frequencies 15- to 50-fold lower than are linear substrates. These results are consistent with mechanisms in which exonuclease or helicase processing of DNA ends permits the forming of recombinants through annealing of complementary single strands. Our data are not consistent with a model involving strand invasion reactions, because such reactions should favor mixtures of linear and circular substrates. We also noted that many of the reaction features seen in vivo were reproduced in a simple in vitro reaction requiring only purified vaccinia virus DNA polymerase, single-strand DNA binding protein, and pairs of linear substrates. The 3′-to-5′ exonuclease activity of poxviral DNA polymerases potentially catalyzes recombination in vivo.

scholarly journals Expanding Diversity of Firmicutes Single-Strand Annealing Proteins: A Putative Role of Bacteriophage-Host Arms Race

2021 ◽  
Vol 12 ◽  
Author(s):  
Kamil Steczkiewicz ◽  
Eric Prestel ◽  
Elena Bidnenko ◽  
Agnieszka K. Szczepankowska
Keyword(s):  
Dna Sequences ◽  
Sequence Similarity ◽  
Reca Protein ◽  
Arms Race ◽  
Single Strand ◽  
Bacterial Phyla ◽  
Single Strand Annealing ◽  
Strand Annealing

Bacteriophage-encoded single strand annealing proteins (SSAPs) are recombinases which can substitute the classical, bacterial RecA and manage the DNA metabolism at different steps of phage propagation. SSAPs have been shown to efficiently promote recombination between short and rather divergent DNA sequences and were exploited for in vivo genetic engineering mainly in Gram-negative bacteria. In opposition to the conserved and almost universal bacterial RecA protein, SSAPs display great sequence diversity. The importance for SSAPs in phage biology and phage-bacteria evolution is underlined by their role as key players in events of horizontal gene transfer (HGT). All of the above provoke a constant interest for the identification and study of new phage recombinase proteins in vivo, in vitro as well as in silico. Despite this, a huge body of putative ssap genes escapes conventional classification, as they are not properly annotated. In this work, we performed a wide-scale identification, classification and analysis of SSAPs encoded by the Firmicutes bacteria and their phages. By using sequence similarity network and gene context analyses, we created a new high quality dataset of phage-related SSAPs, substantially increasing the number of annotated SSAPs. We classified the identified SSAPs into seven distinct families, namely RecA, Gp2.5, RecT/Redβ, Erf, Rad52/22, Sak3, and Sak4, organized into three superfamilies. Analysis of the relationships between the revealed protein clusters led us to recognize Sak3-like proteins as a new distinct SSAP family. Our analysis showed an irregular phylogenetic distribution of ssap genes among different bacterial phyla and specific phages, which can be explained by the high rates of ssap HGT. We propose that the evolution of phage recombinases could be tightly linked to the dissemination of bacterial phage-resistance mechanisms (e.g., abortive infection and CRISPR/Cas systems) targeting ssap genes and be a part of the constant phage-bacteria arms race.

scholarly journals The Yeast Recombinational Repair Protein Rad59 Interacts With Rad52 and Stimulates Single-Strand Annealing

Genetics ◽  
2001 ◽  
Vol 159 (2) ◽  
pp. 515-525 ◽  
Author(s):  
Allison P Davis ◽  
Lorraine S Symington
Keyword(s):  
Protein A ◽  
Single Strand ◽  
Physical Interaction ◽  
Dna Double Strand Breaks ◽  
Single Stranded Dna ◽  
Single Strand Annealing ◽  
Strand Annealing ◽  
Recombination Pathway

Abstract The yeast RAD52 gene is essential for homology-dependent repair of DNA double-strand breaks. In vitro, Rad52 binds to single- and double-stranded DNA and promotes annealing of complementary single-stranded DNA. Genetic studies indicate that the Rad52 and Rad59 proteins act in the same recombination pathway either as a complex or through overlapping functions. Here we demonstrate physical interaction between Rad52 and Rad59 using the yeast two-hybrid system and co-immunoprecipitation from yeast extracts. Purified Rad59 efficiently anneals complementary oligonucleotides and is able to overcome the inhibition to annealing imposed by replication protein A (RPA). Although Rad59 has strand-annealing activity by itself in vitro, this activity is insufficient to promote strand annealing in vivo in the absence of Rad52. The rfa1-D288Y allele partially suppresses the in vivo strand-annealing defect of rad52 mutants, but this is independent of RAD59. These results suggest that in vivo Rad59 is unable to compete with RPA for single-stranded DNA and therefore is unable to promote single-strand annealing. Instead, Rad59 appears to augment the activity of Rad52 in strand annealing.

scholarly journals RecF and RecR Play Critical Roles in the Homologous Recombination and Single-Strand Annealing Pathways of Mycobacteria

2015 ◽  
Vol 197 (19) ◽  
pp. 3121-3132 ◽  
Author(s):  
Richa Gupta ◽  
Stewart Shuman ◽  
Michael S. Glickman
Keyword(s):  
Dna Repair ◽  
Homologous Recombination ◽  
Strand Break ◽  
Single Strand ◽  
Central Position ◽  
End Joining ◽  
Dna Double Strand Break ◽  
Single Strand Annealing ◽  
Strand Annealing

ABSTRACTMycobacteria encode three DNA double-strand break repair pathways: (i) RecA-dependent homologous recombination (HR), (ii) Ku-dependent nonhomologous end joining (NHEJ), and (iii) RecBCD-dependent single-strand annealing (SSA). Mycobacterial HR has two presynaptic pathway options that rely on the helicase-nuclease AdnAB and the strand annealing protein RecO, respectively. Ablation ofadnABorrecOindividually causes partial impairment of HR, but loss ofadnABandrecOin combination abolishes HR. RecO, which can accelerate annealing of single-stranded DNAin vitro, also participates in the SSA pathway. The functions of RecF and RecR, which, in other model bacteria, function in concert with RecO as mediators of RecA loading, have not been examined in mycobacteria. Here, we present a genetic analysis ofrecFandrecRin mycobacterial recombination. We find that RecF, like RecO, participates in the AdnAB-independent arm of the HR pathway and in SSA. In contrast, RecR is required for all HR in mycobacteria and for SSA. The essentiality of RecR as an agent of HR is yet another distinctive feature of mycobacterial DNA repair.IMPORTANCEThis study clarifies the molecular requirements for homologous recombination in mycobacteria. Specifically, we demonstrate that RecF and RecR play important roles in both the RecA-dependent homologous recombination and RecA-independent single-strand annealing pathways. Coupled with our previous findings (R. Gupta, M. Ryzhikov, O. Koroleva, M. Unciuleac, S. Shuman, S. Korolev, and M. S. Glickman, Nucleic Acids Res 41:2284–2295, 2013,http://dx.doi.org/10.1093/nar/gks1298), these results revise our view of mycobacterial recombination and place the RecFOR system in a central position in homology-dependent DNA repair.

scholarly journals Rejoining of DNA double-strand breaks in vitro by single-strand annealing

1998 ◽  
Vol 258 (2) ◽  
pp. 387-395 ◽  
Author(s):  
Bernd Gottlich ◽  
Susanne Reichenberger ◽  
Elke Feldmann ◽  
Petra Pfeiffer
Keyword(s):  
Double Strand Breaks ◽  
Single Strand ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Single Strand Annealing ◽  
Strand Annealing

scholarly journals A Rad51-Independent Pathway Promotes Single-Strand Template Repair in Gene Editing

2020 ◽  
Author(s):  
Danielle N. Gallagher ◽  
Nhung Pham ◽  
Annie M. Tsai ◽  
Abigail N. Janto ◽  
Jihyun Choi ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Gene Editing ◽  
Fold Increase ◽  
Single Strand ◽  
Critical Function ◽  
Strand Breaks ◽  
Independent Events ◽  
Single Strand Annealing ◽  
Strand Annealing

AbstractThe Rad51/RecA family of recombinases perform a critical function in typical repair of double-strand breaks (DSBs): strand invasion of a resected DSB end into a homologous double-stranded DNA (dsDNA) template sequence to facilitate repair. However, repair of a DSB using single stranded DNA (ssDNA) as a template, a common method of CRISPR/Cas9-mediated gene editing, is Rad51-independent. We have analyzed the genetic requirements for these Rad51-independent events in Saccharomyces cerevisiae in two different assays. Gene editing events were carried out either by creating a DSB with the site-specific HO endonuclease and repairing the DSB with 80-nt single-stranded oligonucleotides (ssODNs) or by using a bacterial retron system that produces ssDNA templates in vivo in combination with DSBs created by Cas9. We show that single strand template repair (SSTR), is dependent on Rad52, Rad59, Srs2 and the Mre11-Rad50-Xrs2 (MRX) complex, but not Rad51, Rad54 or Rad55. Srs2 acts to prevent overloading of Rad51 on the ssDNA filament, whereas Rad59 appears to alleviate the inhibition of Rad51 on Rad52’s strand annealing activity; thus, deletion of RAD51 suppresses both the srs2Δ and rad59Δ phenotypes. This same suppression by rad51Δ of rad59Δ is found in another DSB repair pathway, single strand annealing (SSA). In contrast, gene targeting using an 80-bp dsDNA template of the same sequence is Rad51-dependent. We also examined SSTR events in which the ssODN carried several mismatches. In the absence of the mismatch repair protein, Msh2, we found that the fate of mismatches carried on the ssDNA template are very different at the 3’ end, which can anneal directly to the resected DSB end, compared to the 5’ end. We also find that DNA polymerase Polδ’s 3’ to 5’ proofreading activity frequently excises a mismatch close to the 3’ end of the template, similar to its removal of heterologies close to the 3’ invading end of the DSB. We further report that SSTR is accompanied by a 600-fold increase in mutations in a region adjacent to the sequences directly undergoing repair. These DNA polymerase ζ-dependent mutations may compromise the accuracy of gene editing.

scholarly journals Examining a DNA Replication Requirement for Bacteriophage λ Red- and Rac Prophage RecET-Promoted Recombination inEscherichia coli

mBio ◽  
2016 ◽  
Vol 7 (5) ◽  
Author(s):  
Lynn C. Thomason ◽  
Nina Costantino ◽  
Donald L. Court
Keyword(s):  
Dna Replication ◽  
Homologous Recombination ◽  
Genetic Engineering ◽  
Single Strand ◽  
Genetic Modifications ◽  
Single Strand Annealing ◽  
Strand Invasion ◽  
Strand Annealing ◽  
Λ Red

ABSTRACTRecombineering,in vivogenetic engineering with bacteriophage homologous recombination systems, is a powerful technique for making genetic modifications in bacteria. Two systems widely used inEscherichia coliare the Red system from phage λ and RecET from the defective Rac prophage. We investigated thein vivodependence of recombineering on DNA replication of the recombining substrate using plasmid targets. For λ Red recombination, when DNA replication of a circular target plasmid is prevented, recombination with single-stranded DNA oligonucleotides is greatly reduced compared to that under replicating conditions. For RecET recombination, when DNA replication of the targeted plasmid is prevented, the recombination frequency is also reduced, to a level identical to that seen for the Red system in the absence of replication. The very low level of oligonucleotide recombination observed in the absence of any phage recombination functions is the same in the presence or absence of DNA replication. In contrast, both the Red and RecET systems recombine a nonreplicating linear dimer plasmid with high efficiency to yield a circular monomer. Therefore, the DNA replication requirement is substrate dependent. Our data are consistent with recombination by both the Red and RecET systems occurring predominately by single-strand annealing rather than by strand invasion.IMPORTANCEBacteriophage homologous recombination systems are widely used forin vivogenetic engineering in bacteria. Single- or double-stranded linear DNA substrates containing short flanking homologies to chromosome targets are used to generate precise and accurate genetic modifications when introduced into bacteria expressing phage recombinases. Understanding the molecular mechanism of these recombination systems will facilitate improvements in the technology. Here, two phage-specific systems are shown to require exposure of complementary single-strand homologous targets for efficient recombination; these single-strand regions may be created during DNA replication or by single-strand exonuclease digestion of linear duplex DNA. Previously,in vitrostudies reported that these recombinases promote the single-strand annealing of two complementary DNAs and also strand invasion of a single DNA strand into duplex DNA to create a three-stranded region. Here,in vivoexperiments show that recombinase-mediated annealing of complementary single-stranded DNA is the predominant recombination pathway inE. coli.

scholarly journals Role of reciprocal exchange, one-ended invasion crossover and single-strand annealing on inverted and direct repeat recombination in yeast: different requirements for the RAD1, RAD10, and RAD52 genes.

Genetics ◽  
1995 ◽  
Vol 139 (1) ◽  
pp. 109-123 ◽  
Author(s):  
F Prado ◽  
A Aguilera
Keyword(s):  
Direct Repeat ◽  
Repeat Sequence ◽  
Single Strand ◽  
Dna Repeats ◽  
Recombination Mechanism ◽  
Reciprocal Exchange ◽  
Independent Events ◽  
Single Strand Annealing ◽  
Strand Annealing

Abstract We have constructed novel DNA substrates (one inverted and three direct repeats) based on the same 0.6-kb repeat sequence to study deletions and inversions in Saccharomyces cerevisiae. Spontaneous deletions occur six to eight times more frequently than inversions, irrespective of the distance between the repeats. This difference can be explained by the observation that deletion events can be mediated by a recombination mechanism that can initiate within the intervening sequence of the repeats. Spontaneous and double-strand break (DSB)-induced deletions occur as RAD52-dependent and RAD52-independent events. Those deletion events initiated through a DSB in the unique intervening sequence require the Rad1/Rad10 endonuclease only if the break is distantly located from the flanking DNA repeats. We propose that deletions can occur as three types of recombination events: the conservative RAD52-dependent reciprocal exchange and the nonconservative events, one-ended invasion crossover, and single-strand annealing (SSA). We suggest that one-ended invasion is RAD52 dependent, whereas SSA is RAD52 independent. Whereas deletions, like inversions, occur through reciprocal exchange, deletions can also occur through SSA or one-ended invasion. We propose that the contribution of reciprocal exchange and one-ended invasion crossover vs. SSA events to overall spontaneous deletions is a feature specific for each repeat system, determined by the initiation event and the availability of the Rad52 protein. We discuss the role of the Rad1/Rad10 endonuclease on the initial steps of one-ended invasion crossover and SSA as a function of the location of the initiation event relative to the repeats. We also show that the frequency of recombination between repeats is the same independent of their location (whether on circular plasmids, linear minichromosomes, or natural chromosomes) and have similar RAD52 dependence.

scholarly journals Neutrophil subtypes shape HIV-specific CD8 T-cell responses after vaccinia virus infection

npj Vaccines ◽  
2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Mauro Di Pilato ◽  
Miguel Palomino-Segura ◽  
Ernesto Mejías-Pérez ◽  
Carmen E. Gómez ◽  
Andrea Rubio-Ponce ◽  
...  
Keyword(s):  
T Cell ◽  
Immune Responses ◽  
Vaccinia Virus ◽  
Adaptive Immune System ◽  
T Cell Responses ◽  
Cd8 T Cell ◽  
Cell Responses ◽  
Adaptive Immune

AbstractNeutrophils are innate immune cells involved in the elimination of pathogens and can also induce adaptive immune responses. Nα and Nβ neutrophils have been described with distinct in vitro capacity to generate antigen-specific CD8 T-cell responses. However, how these cell types exert their role in vivo and how manipulation of Nβ/Nα ratio influences vaccine-mediated immune responses are not known. In this study, we find that these neutrophil subtypes show distinct migratory and motility patterns and different ability to interact with CD8 T cells in the spleen following vaccinia virus (VACV) infection. Moreover, after analysis of adhesion, inflammatory, and migration markers, we observe that Nβ neutrophils overexpress the α4β1 integrin compared to Nα. Finally, by inhibiting α4β1 integrin, we increase the Nβ/Nα ratio and enhance CD8 T-cell responses to HIV VACV-delivered antigens. These findings provide significant advancements in the comprehension of neutrophil-based control of adaptive immune system and their relevance in vaccine design.

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