RecA protein-dependent R-loop formation in vitro

Kasahara, Megumi; Clikeman, Jennifer A.; Bates, David; Kogoma, Tokio

doi:10.1101/gad.14.3.360

Cited by 64 publications

(14 citation statements)

References 28 publications

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“…Our observation that ONs with a LNA or 2′-OMe RNA backbone can hybridize to the outgoing strand of a short synaptic complex suggests that transcripts in the cell could participate in a similar reaction and that the hybridized RNA could serve as a primer for the initiation of DNA synthesis. This pathway for RNA uptake is distinct from the recently described inverse strand exchange reaction (19,20). In that reaction, dsDNA in a RecA or Rad51 filament strand exchanges with a homologous single-stranded DNA or RNA.…”

Section: Discussionmentioning

confidence: 86%

“…The extended structure of dsDNA that is bound to a recombinase facilitates several unconventional strand exchange reactions. For example, the inverse of regular strand exchange occurs when dsDNA occupies the primary binding site and ssDNA or ssRNA occupies the secondary binding site of a RecA or Rad51 filament ( − ). If dsDNAs occupy both binding sites of the filament, reciprocal four-strand exchange between the two duplexes can sometimes be observed ( , ).…”

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confidence: 99%

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The Synaptic Complex of RecA Protein Participates in Hybridization and Inverse Strand Exchange Reactions

Gamper

Nulf

Corey³

et al. 2003

Biochemistry

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RecA protein catalyzes strand exchange between homologous single-stranded and double-stranded DNAs. In the presence of ATPgammaS, the post-strand exchange synaptic complex is a stable end product that can be studied. Here we ask whether such complexes can hybridize to or exchange with DNA, 2'-OMe RNA, PNA, or LNA oligonucleotides. Using a gel mobility shift assay, we show that the displaced strand of a 45 bp synaptic complex can hybridize to complementary oligonucleotides with different backbones to form a four-stranded (double D-loop) joint that survives removal of the RecA protein. This hybridization reaction, which confirms the single-stranded character of the displaced strand in a synaptic complex, might initiate recombination-dependent DNA replication if it occurs in vivo. We also show that either strand of the heteroduplex in a 30 bp synaptic complex can be replaced with a homologous DNA oligonucleotide in a strand exchange reaction that is mediated by the RecA filament. Consistent with the important role that deoxyribose plays in strand exchange, oligonucleotides with non-DNA backbones did not participate in this reaction. The hybridization and strand exchange reactions reported here demonstrate that short synaptic complexes are dynamic structures even in the presence of ATPgammaS.

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Section: Discussionmentioning

confidence: 86%

mentioning

confidence: 99%

The Synaptic Complex of RecA Protein Participates in Hybridization and Inverse Strand Exchange Reactions

Gamper

Nulf

Corey³

et al. 2003

Biochemistry

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“…Accumulating genetic evidence in E. coli has suggested that one form of recombination-dependent replication known as constitutive stable DNA replication is achieved by RecAfacilitated joint molecule formation between an RNA transcript and the bacterial chromosome (49). Biochemical support for this idea comes from studies showing RecA can promote annealing of an RNA oligonucleotide with a free single-stranded DNA or with a preformed single-stranded loop in a DNA heteroduplex, (50) and furthermore, can perform an inverse strand exchange reaction between a DNA heteroduplex and a homologous single-stranded RNA (51,52). All of these pairing reactions performed with RNA are much less efficient than with DNA, presumably due to steric hindrance encountered during protein-promoted extension of the RNA phosphodiester backbone in preparing for intermolecular association (53).…”

Section: Discussionmentioning

confidence: 99%

A RecA Homologue in Ustilago maydis That Is Distinct and Evolutionarily Distant from Rad51 Actively Promotes DNA Pairing Reactions in the Absence of Auxiliary Factors

Bennett

Holloman

2001

Biochemistry

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Two RecA homologues have been identified to date in Ustilago maydis. One is orthologous to Rad51 while the other, Rec2, is structurally quite divergent and evolutionarily distant. DNA repair and recombination proficiency in U. maydis requires both Rec2 and Rad51. Here we have examined biochemical activities of Rec2 protein purified after overexpression of the cloned gene. Rec2 requires DNA as a cofactor to hydrolyze ATP and depends on ATP to promote homologous pairing and DNA strand exchange. ATPgammaS was found to substitute for ATP in all pairing reactions examined. With superhelical DNA and a homologous single-stranded oligonucleotide as substrates, Rec2 actively promoted formation and dissociation of D-loops. When an RNA oligonucleotide was substituted it was found that R-loops could also be formed and utilized as primer/template for limited DNA synthesis. In DNA strand exchange reactions using oligonucleotides, we found that Rec2 exhibited a pairing bias that is opposite that of RecA. Single-stranded oligonucleotides were activated for DNA strand exchange when attached as tails protruding from a duplex sequence due to enhanced binding of Rec2. The results indicate that Rec2 is competent, and in certain ways even better than Rad51, in the ability to provide the fundamental DNA pairing activity necessary for recombinational repair. We propose that the emerging paradigm for homologous recombination featuring Rad51 as the essential catalytic component for strand exchange may not be universal in eukaryotes.

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“…Previous work showed that the RecA recombinase of Escherichia coli can promote formation of DNA−RNA hybrids. 257,258 Yeast RNA-templated DSB repair is strongly dependent on the recombinase Rad52, a fundamental protein in DNA repair by HR. 169,259 However, knockout of the RAD52 gene, while reducing the frequency of DSB repair by RNA by a factor of 10, does not eliminate DSB repair by RNA, indicating that Rad52-independent RNA-templated DSB repair mechanisms do exist.…”

Section: How Does Rna-templated Dsb Repair Work?mentioning

confidence: 99%

“…Instead, the sensitivity to RNase H activity indicates that DNA–RNA hybrids must form to transfer information from RNA to DNA. Previous work showed that the RecA recombinase of Escherichia coli can promote formation of DNA–RNA hybrids. , Yeast RNA-templated DSB repair is strongly dependent on the recombinase Rad52, a fundamental protein in DNA repair by HR. , However, knockout of the RAD52 gene, while reducing the frequency of DSB repair by RNA by a factor of 10, does not eliminate DSB repair by RNA, indicating that Rad52-independent RNA-templated DSB repair mechanisms do exist. These results in yeast are supported by in vitro experiments corroborating the ability of the Rad52 protein to catalyze the annealing of RNA to DNA .…”

Section: Rna-templated Dna Repair In Yeast and Mammalsmentioning

confidence: 99%

From “Cellular” RNA to “Smart” RNA: Multiple Roles of RNA in Genome Stability and Beyond

et al. 2018

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Coding for proteins has been considered the main function of RNA since the "central dogma" of biology was proposed. The discovery of noncoding transcripts shed light on additional roles of RNA, ranging from the support of polypeptide synthesis, to the assembly of subnuclear structures, to gene expression modulation. Cellular RNA has therefore been recognized as a central player in often unanticipated biological processes, including genomic stability. This ever-expanding list of functions inspired us to think of RNA as a "smart" phone, which has replaced the older obsolete "cellular" phone. In this review, we summarize the last two decades of advances in research on the interface between RNA biology and genome stability. We start with an account of the emergence of noncoding RNA, and then we discuss the involvement of RNA in DNA damage signaling and repair, telomere maintenance, and genomic rearrangements. We continue with the depiction of single-molecule RNA detection techniques, and we conclude by illustrating the possibilities of RNA modulation in hopes of creating or improving new therapies. The widespread biological functions of RNA have made this molecule a reoccurring theme in basic and translational research, warranting it the transcendence from classically studied "cellular" RNA to "smart" RNA.

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RecA protein-dependent R-loop formation in vitro

Cited by 64 publications

References 28 publications

The Synaptic Complex of RecA Protein Participates in Hybridization and Inverse Strand Exchange Reactions

The Synaptic Complex of RecA Protein Participates in Hybridization and Inverse Strand Exchange Reactions

A RecA Homologue in Ustilago maydis That Is Distinct and Evolutionarily Distant from Rad51 Actively Promotes DNA Pairing Reactions in the Absence of Auxiliary Factors

From “Cellular” RNA to “Smart” RNA: Multiple Roles of RNA in Genome Stability and Beyond

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