scholarly journals Bacterial RecA Protein Promotes Adenoviral Recombination duringIn VitroInfection

mSphere ◽  
2018 ◽  
Vol 3 (3) ◽  
Author(s):  
Jeong Yoon Lee ◽  
Ji Sun Lee ◽  
Emma C. Materne ◽  
Rahul Rajala ◽  
Ashrafali M. Ismail ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Hot Spots ◽  
Genetic Recombination ◽  
Reca Protein ◽  
Human Adenovirus ◽  
Penton Base ◽  
Content Type ◽  
E Coli ◽  
Recombination Hot Spots ◽  
Loop 2

ABSTRACTAdenovirus infections in humans are common and sometimes lethal. Adenovirus-derived vectors are also commonly chosen for gene therapy in human clinical trials. We have shown in previous work that homologous recombination between adenoviral genomes of human adenovirus species D (HAdV-D), the largest and fastest growing HAdV species, is responsible for the rapid evolution of this species. Because adenovirus infection initiates in mucosal epithelia, particularly at the gastrointestinal, respiratory, genitourinary, and ocular surfaces, we sought to determine a possible role for mucosal microbiota in adenovirus genome diversity. By analysis of known recombination hot spots across 38 human adenovirus genomes in species D (HAdV-D), we identified nucleotide sequence motifs similar to bacterial Chi sequences, which facilitate homologous recombination in the presence of bacterial Rec enzymes. These motifs, referred to here as ChiAD, were identified immediately 5′ to the sequence encoding penton base hypervariable loop 2, which expresses the arginine-glycine-aspartate moiety critical to adenoviral cellular entry. Coinfection with two HAdV-Ds in the presence of anEscherichia colilysate increased recombination; this was blocked in a RecA mutant strain,E. coliDH5α, or upon RecA depletion. Recombination increased in the presence ofE. colilysate despite a general reduction in viral replication. RecA colocalized with viral DNA in HAdV-D-infected cell nuclei and was shown to bind specifically to ChiADsequences. These results indicate that adenoviruses may repurpose bacterial recombination machinery, a sharing of evolutionary mechanisms across a diverse microbiota, and unique example of viral commensalism.IMPORTANCEAdenoviruses are common human mucosal pathogens of the gastrointestinal, respiratory, and genitourinary tracts and ocular surface. Here, we report finding Chi-like sequences in adenovirus recombination hot spots. Adenovirus coinfection in the presence of bacterial RecA protein facilitated homologous recombination between viruses. Genetic recombination led to evolution of an important external feature on the adenoviral capsid, namely, the penton base protein hypervariable loop 2, which contains the arginine-glycine-aspartic acid motif critical to viral internalization. We speculate that free Rec proteins present in gastrointestinal secretions upon bacterial cell death facilitate the evolution of human adenoviruses through homologous recombination, an example of viral commensalism and the complexity of virus-host interactions, including regional microbiota.

Electron Microscopy and image analysis of nucleo-protein complexes

1994 ◽  
Vol 52 ◽  
pp. 88-89
Author(s):  
E. H. Egelman ◽  
X. Yu
Keyword(s):  
Electron Microscopy ◽  
Image Analysis ◽  
Normal Cell ◽  
Genetic Recombination ◽  
Protein Complexes ◽  
Chromosomal Rearrangements ◽  
Reca Protein ◽  
E Coli ◽  
Helical Polymer ◽  
Specific Protease

The RecA protein of E. coli has been shown to mediate genetic recombination, regulate its own synthesis, control the expression of other genes, act as a specific protease, form a helical polymer and have an ATPase activity, among other observed properties. The unusual filament formed by the RecA protein on DNA has not previously been shown to exist outside of bacteria. Within this filament, the 36 Å pitch of B-form DNA is extended to about 95 Å, the pitch of the RecA helix. We have now establishedthat similar nucleo-protein complexes are formed by bacteriophage and yeast proteins, and availableevidence suggests that this structure is universal across all of biology, including humans. Thus, understanding the function of the RecA protein will reveal basic mechanisms, in existence inall organisms, that are at the foundation of general genetic recombination and repair.Recombination at this moment is assuming an importance far greater than just pure biology. The association between chromosomal rearrangements and neoplasms has become stronger and stronger, and these rearrangements are most likely products of the recombinatory apparatus of the normal cell. Further, damage to DNA appears to be a major cause of cancer.

scholarly journals Sequence Analysis of Tn10 Insertion Sites in a Collection of Escherichia coli Strains Used for Genetic Mapping and Strain Construction

1998 ◽  
Vol 180 (23) ◽  
pp. 6408-6411 ◽  
Author(s):  
Brian P. Nichols ◽  
Obaid Shafiq ◽  
Victoria Meiners
Keyword(s):  
Escherichia Coli ◽  
Sequence Analysis ◽  
Hot Spots ◽  
Insertion Site ◽  
Inverse Pcr ◽  
E Coli ◽  
Chromosome Sequence ◽  
Tn10 Insertion ◽  
Insertion Sites ◽  
Recombination Hot Spots

ABSTRACT The chromosomal insertion sites of Tn10-containingEscherichia coli strains were amplified by inverse PCR, and the nucleotide sequences of the junctions were determined. In 95 strains analyzed, 88 unique Tn10 positions were determined and matched to the E. coli chromosome sequence. Two gaps in insertion site positions were noted, one including the terminus of DNA replication and another bounded by recombination hot spots RhsA and RhsB.

scholarly journals Frequent Homologous Recombination Events between Molecules of One RNA Component in a Multipartite RNA Virus

2000 ◽  
Vol 74 (9) ◽  
pp. 4214-4219 ◽  
Author(s):  
A. Bruyere ◽  
M. Wantroba ◽  
S. Flasinski ◽  
A. Dzianott ◽  
J. J. Bujarski
Keyword(s):  
Homologous Recombination ◽  
Hot Spots ◽  
Rna Virus ◽  
Hot Spot ◽  
Rna Replication ◽  
Rna Recombination ◽  
Brome Mosaic Bromovirus ◽  
Local Lesion Host ◽  
Minor Sequence ◽  
Recombination Hot Spots

ABSTRACT Brome mosaic bromovirus (BMV), a tripartite plus-sense RNA virus, has been used as a model system to study homologous RNA recombination among molecules of the same RNA component. Pairs of BMV RNA3 variants carrying marker mutations at different locations were coinoculated on a local lesion host, and the progeny RNA3 in a large number of lesions was analyzed. The majority of doubly infected lesions accumulated the RNA3 recombinants. The distribution of the recombinant types was relatively even, indicating that both RNA3 counterparts could serve as donor or as acceptor molecules. The frequency of crossovers between one pair of RNA3 variants, which possessed closely located markers, was similar to that of another pair of RNA3 variants with more distant markers, suggesting the existence of an internal recombination hot spot. The majority of crossovers were precise, but some recombinants had minor sequence modifications, possibly marking the sites of imprecise homologous crossovers. Our results suggest discontinuous RNA replication, with the replicase changing among the homologous RNA templates and generating RNA diversity. This approach can be easily extended to other RNA viruses for identification of homologous recombination hot spots.

RecA-DNA complexes: What we can learn about structure and dynamics from disordered filaments using computed image analysis

1986 ◽  
Vol 44 ◽  
pp. 144-145
Author(s):  
E.H. Egelman
Keyword(s):  
Image Analysis ◽  
Genetic Recombination ◽  
Reca Protein ◽  
Strand Transfer ◽  
Homologous Pairing ◽  
Dna Complexes ◽  
Helical Polymers ◽  
E Coli ◽  
Structure And Dynamics ◽  
Static Images

The recA protein (38,000MW) of E. coli forms helical polymers which are able, in an ATP-dependent reaction, to mediate the entire genetic recombination process, including the search for homology, homologous pairing, and strand transfer. We have been using computed image analysis of electron micrographs of different recA complexes in an effort to understand the function of this protein in recombination. These filaments typically show poor helical order. We have studied the systematic deviations from helical order (the disorder) present in static images of recA complexes as a means of understanding the dynamics of recA filaments in solution.

scholarly journals Genomic characterization of a novel human adenovirus type 31 recombinant in the hexon gene

2011 ◽  
Vol 92 (12) ◽  
pp. 2770-2775 ◽  
Author(s):  
Yuki Matsushima ◽  
Hideaki Shimizu ◽  
Tung Gia Phan ◽  
Hiroshi Ushijima
Keyword(s):  
Acute Gastroenteritis ◽  
Recombinant Adenovirus ◽  
Genetic Recombination ◽  
Human Adenovirus ◽  
Adenovirus Type ◽  
Close Relative ◽  
Hexon Gene ◽  
Penton Base ◽  
Genomic Characterization

A novel human recombinant adenovirus of species A (HAdV-A31 MZ) was isolated from a patient with acute gastroenteritis in Japan. The complete genome of HAdV-A31 strain MZ contains 33 776 bp. Analysis of the hexon gene of HAdV-A31 MZ indicated that its hexon sequence is the result of a genetic recombination between those of HAdV-A31 and a close relative to HAdV-A12. The recombination sites were found around the border of hypervariable loops 1 and 2 in the hexon gene, which are the most important determinants for virus neutralization. Loops 1 and 2 of this virus were genetically related to HAdV-A12, whereas all other parts of the genome were highly similar to HAdV-A31. In order to understand the evolution of adenoviruses correctly and to avoid misidentification of HAdV types, we recommend characterizing not only the hexon gene, but also the penton base and fiber genes.

scholarly journals Genetic Functions Promoting Homologous Recombination in Escherichia coli: A Study of Inversions in Phage Λ

Genetics ◽  
1987 ◽  
Vol 115 (1) ◽  
pp. 11-24
Author(s):  
Don G Ennis ◽  
Susan K Amundsen ◽  
Gerald R Smith
Keyword(s):  
Escherichia Coli ◽  
Homologous Recombination ◽  
Molecular Model ◽  
Reca Protein ◽  
Dna Polymerase I ◽  
E Coli ◽  
The Right ◽  
Polymerase I ◽  
Recbcd Enzyme ◽  
A Current

ABSTRACT We have studied homologous recombination in a derivative of phage λ containing two 1.4-kb repeats in inverted orientation. Inversion of the intervening 2.5-kb segment occurred efficiently by the Escherichia coli RecBC pathway but markedly less efficiently by the λ Red pathway or the E. coli RecE or RecF pathways. Inversion by the RecBCD pathway was stimulated by Chi sites located to the right of the invertible segment; this stimulation decreased exponentially by a factor of about 2 for each 2.2 kb between the invertible segment and the Chi site. In addition to RecA protein and RecBCD enzyme, inversion by the RecBC pathway required single-stranded DNA binding protein, DNA gyrase, DNA polymerase I and DNA ligase. Inversion appeared to occur either intra- or intermolecularly. These results are discussed in the framework of a current molecular model for the RecBC pathway of homologous recombination.

scholarly journals Gene Disruption through Homologous Recombination inSpiroplasma citri: an scm1-Disrupted Motility Mutant Is Pathogenic

1999 ◽  
Vol 181 (24) ◽  
pp. 7449-7456 ◽  
Author(s):  
Sybille Duret ◽  
Jean-Luc Danet ◽  
Monique Garnier ◽  
Joël Renaudin
Keyword(s):  
Homologous Recombination ◽  
Gene Disruption ◽  
Reca Protein ◽  
Specific Gene ◽  
Gene Fragment ◽  
Wild Type ◽  
Spiroplasma Citri ◽  
E Coli ◽  
Circulifer Haematoceps ◽  
Motility Mutant

ABSTRACT To determine whether homologous recombination could be used to inactivate selected genes in Spiroplasma citri, plasmid constructs were designed to disrupt the motility gene scm1. An internal scm1 gene fragment was inserted into plasmid pKT1, which replicates in Escherichia coli but not inS. citri, and into the S. citri oriC plasmid pBOT1, which replicates in spiroplasma cells as well as in E. coli. Electrotransformation of S. citri with the nonreplicative, recombinant plasmid pKTM1 yielded no transformants. In contrast, spiroplasmal transformants were obtained with the replicative, pBOT1-derived plasmid pCJ32. During passaging of the transformants, the plasmid was found to integrate into the chromosome by homologous recombination either at the oriC region or at the scm1 gene. In the latter case, plasmid integration by a single crossover between the scm1 gene fragment carried by the plasmid and the full-length scm1 gene carried by the chromosome led to a nonmotile phenotype. Transmission of thescm1-disrupted mutant to periwinkle (Catharanthus roseus) plants through injection into the leafhopper vector (Circulifer haematoceps) showed that the motility mutant multiplied in the insects and was efficiently transmitted to plants, in which it induced symptoms similarly to the wild-type S. citri strain. These results suggest that the spiroplasmal motility may not be essential for pathogenicity and that, more broadly, the S. citri oriC plasmids can be considered promising tools for specific gene disruption by promoting homologous recombination in S. citri, a mollicute which probably lacks a functional RecA protein.

scholarly journals Predicting the Next Eye Pathogen: Analysis of a Novel Adenovirus

mBio ◽  
2013 ◽  
Vol 4 (2) ◽  
Author(s):  
Christopher M. Robinson ◽  
Xiaohong Zhou ◽  
Jaya Rajaiya ◽  
Mohammad A. Yousuf ◽  
Gurdeep Singh ◽  
...  
Keyword(s):  
Systems Biology ◽  
Genetic Recombination ◽  
Human Adenovirus ◽  
Adenovirus Type ◽  
Genetic Alterations ◽  
Laboratory Simulation ◽  
Penton Base ◽  
Emerging Viruses ◽  
Evolutionary Mechanisms

ABSTRACTFor DNA viruses, genetic recombination, addition, and deletion represent important evolutionary mechanisms. Since these genetic alterations can lead to new, possibly severe pathogens, we applied a systems biology approach to study the pathogenicity of a novel human adenovirus with a naturally occurring deletion of the canonical penton base Arg-Gly-Asp (RGD) loop, thought to be critical to cellular entry by adenoviruses. Bioinformatic analysis revealed a new highly recombinant species D human adenovirus (HAdV-D60). A synthesis ofin silicoand laboratory approaches revealed a potential ocular tropism for the new virus.In vivo, inflammation induced by the virus was dramatically greater than that by adenovirus type 37, a major eye pathogen, possibly due to a novel alternate ligand, Tyr-Gly-Asp (YGD), on the penton base protein. The combination of bioinformatics and laboratory simulation may have important applications in the prediction of tissue tropism for newly discovered and emerging viruses.IMPORTANCEThe ongoing dance between a virus and its host distinctly shapes how the virus evolves. While human adenoviruses typically cause mild infections, recent reports have described newly characterized adenoviruses that cause severe, sometimes fatal human infections. Here, we report a systems biology approach to show how evolution has affected the disease potential of a recently identified novel human adenovirus. A comprehensive understanding of viral evolution and pathogenicity is essential to our capacity to foretell the potential impact on human disease for new and emerging viruses.

scholarly journals Role of Homologous Recombination in Adaptive Diversification of Extraintestinal Escherichia coli

2012 ◽  
Vol 195 (2) ◽  
pp. 231-242 ◽  
Author(s):  
Sandip Paul ◽  
Elena V. Linardopoulou ◽  
Mariya Billig ◽  
Veronika Tchesnokova ◽  
Lance B. Price ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Homologous Recombination ◽  
Bacterial Pathogens ◽  
Genomic Diversity ◽  
Fluoroquinolone Resistance ◽  
Content Type ◽  
Adaptive Diversification ◽  
E Coli ◽  
And Function ◽  
Core Genes

ABSTRACTThe contribution of homologous exchange (recombination) of core genes in the adaptive evolution of bacterial pathogens is not well understood. To investigate this, we analyzed fully assembled genomes of twoEscherichia colistrains from sequence type 131 (ST131), a clonal group that is both the leading cause of extraintestinalE. coliinfections and the main source of fluoroquinolone-resistantE. coli. Although the sequences of each of the seven multilocus sequence typing genes were identical in the two ST131 isolates, the strains diverged from one another by homologous recombination that affected at least 9% of core genes. This was on a par with the contribution to genomic diversity of horizontal gene transfer and point gene mutation. The genomic positions of recombinant and mobile genetic regions were partially linked, suggesting their concurrent occurrence. One of the genes affected by homologous recombination wasfimH, which encodes mannose-specific type 1 fimbrial adhesin, resulting in functionally distinct copies of the gene in ST131 strains. One strain, a uropathogenic isolate, had a pathoadaptive variant offimHthat was acquired by homologous replacement into the commensal strain background. Close examination of FimH structure and function in additional ST131 isolates revealed that recombination led to acquisition of several functionally distinct variants that, upon homologous exchange, were targeted by a variety of pathoadaptive mutations under strong positive selection. Different recombinantfimHstrains also showed a strong clonal association with ST131 isolates that had distinct fluoroquinolone resistance profiles. Thus, homologous recombination of core genes plays a significant role in adaptive diversification of bacterial pathogens, especially at the level of clonally related groups of isolates.

scholarly journals Silencing Homologous RNA Recombination Hot Spots with GC-Rich Sequences in Brome Mosaic Virus

1998 ◽  
Vol 72 (2) ◽  
pp. 1122-1130 ◽  
Author(s):  
Peter D. Nagy ◽  
Jozef J. Bujarski
Keyword(s):  
Homologous Recombination ◽  
Mosaic Virus ◽  
Hot Spots ◽  
Rna Virus ◽  
Hot Spot ◽  
Brome Mosaic Virus ◽  
Complex Nature ◽  
Template Switching ◽  
Rna Recombination ◽  
Recombination Hot Spots

ABSTRACT It has been observed that AU-rich sequences form homologous recombination hot spots in brome mosaic virus (BMV), a tripartite positive-stranded RNA virus of plants (P. D. Nagy and J. J. Bujarski, J. Virol. 71:3799–3810, 1997). To study the effect of GC-rich sequences on the recombination hot spots, we inserted 30-nucleotide-long GC-rich sequences downstream of AU-rich homologous recombination hot spot regions in parental BMV RNAs (RNA2 and RNA3). Although these insertions doubled the length of sequence identity in RNA2 and RNA3, the incidence of homologous RNA2 and RNA3 recombination was reduced markedly. Four different, both highly structured and nonstructured downstream GC-rich sequences had a similar “homologous recombination silencing” effect on the nearby hot spots. The GC-rich sequence-mediated recombination silencing mapped to RNA2, as it was observed when the GC-rich sequence was inserted at downstream locations in both RNA2 and RNA3 or only in the RNA2 component. On the contrary, when the downstream GC-rich sequence was present only in the RNA3 component, it increased the incidence of homologous recombination. In addition, upstream insertions of similar GC-rich sequences increased the incidence of homologous recombination within downstream hot spot regions. Overall, this study reveals the complex nature of homologous recombination in BMV, where sequences flanking the common hot spot regions affect recombination frequency. A replicase-driven template-switching model is presented to explain recombination silencing by GC-rich sequences.

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