DNA Strand Exchange Mediated by the <i>Escherichia</i> <i>coli</i> RecA Protein Initiates in the Minor Groove of Double-Stranded DNA

Zhou, Xiaohua; Adzuma, Kenji

doi:10.1021/bi9630063

Cited by 42 publications

(31 citation statements)

References 40 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Taken together, data obtained from a variety of biochemical methods, including chemical modification, 21,22 base analog perturbation, 15,19,23 affinity modification, 20,24,25 topology testing, 26,27 fluorescence, 28,29 gel mobility shift, 30 radioprobing, 31 and interfluorophore distance measurements, 32 support the conclusion that homology recognition leading to the observed post-strand-exchange intermediate occurs via minor groove contacts with the substrate dsDNA. It must be emphasized that the mechanistic conclusion is inferred from the steadystate structure of an intermediate after strand exchange.…”

Section: Introductionmentioning

confidence: 63%

“…[15][16][17][18][19] In the latter model, however, the target dsDNA is at least partially melted before pairing and then the homologous sequences are recognized by forming regular Watson-Crick interactions between the complementary strand of the duplex DNA and the incoming ssDNA. 20,21 Importantly, the two models require different mechanistic strategies and kinetics.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Construction and evaluation of a kinetic scheme for RecA‐mediated DNA strand exchange

2006

View full text Add to dashboard Cite

The Escherichia coli RecA protein is the prototype of a class of proteins playing a central role in genomic repair and recombination in all organisms. The unresolved mechanistic strategy by which RecA aligns a single strand of DNA with a duplex DNA and mediates a DNA strand switch is central to understanding its recombinational activities. Toward a molecular-level understanding of RecA-mediated DNA strand exchange, we explored its mechanism using oligonucleotide substrates and the intrinsic fluorescence of 6-methylisoxanthopterin (6MI). Steady- and presteady-state spectrofluorometric data demonstrate that the reaction proceeds via a sequential four-step mechanism comprising a rapid, bimolecular association step followed by three slower unimolecular steps. Previous authors have proposed multistep mechanisms involving two or three steps. Careful analysis of the differences among the experimental systems revealed a previously undiscovered intermediate (N1) whose formation may be crucial in the kinetic discrimination of homologous and heterologous sequences. This observation has important implications for probing the fastest events in DNA strand exchange using 6MI to further elucidate the molecular mechanisms of recombination and recombinational repair.

show abstract

Section: Introductionmentioning

confidence: 63%

Section: Introductionmentioning

confidence: 99%

Construction and evaluation of a kinetic scheme for RecA‐mediated DNA strand exchange

2006

View full text Add to dashboard Cite

show abstract

“…The question of whether the single-stranded DNA-RecA filament binds to the major or minor groove of duplex DNA is still controversial (48,49,(51)(52)(53)(54)(55)(56). In our description, we have assumed that it binds to the minor groove, but major groove binding is also possible with our model.…”

Section: Discussionmentioning

confidence: 99%

“…Homologous alignment of singlestranded DNA and double-stranded DNA is potentially explained by triplex formation in which, by forming additional base pair specific hydrogen bonds, the single strand is placed in the major groove of the double-stranded DNA with the identical strands in a parallel orientation (47)(48)(49)(50). Some reports described observations favorable to the initial interaction from the minor groove (51)(52)(53)(54)(55)(56), but the advantage to homologous pairing has not been clarified to date.…”

mentioning

confidence: 99%

Base pair switching by interconversion of sugar puckers in DNA extended by proteins of RecA-family: A model for homology search in homologous genetic recombination

Nishinaka

Shinohara

Ito

et al. 1998

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

View full text Add to dashboard Cite

Escherichia coli RecA is a representative of proteins from the RecA family, which promote homologous pairing and strand exchange between double-stranded DNA and single-stranded DNA. These reactions are essential for homologous genetic recombination in various organisms. From NMR studies, we previously reported a novel deoxyribose-base stacking interaction between adjacent residues on the extended singlestranded DNA bound to RecA protein. In this study, we found that the same DNA structure was induced by the binding to Saccharomyces cerevisiae Rad51 protein, indicating that the unique DNA structure induced by the binding to RecA-homologs was conserved from prokaryotes to eukaryotes. On the basis of this structure, we have formulated the structure of duplex DNA within filaments formed by RecA protein and its homologs. Two types of molecular structures are presented. One is the duplex structure that has the N-type sugar pucker. Its helical pitch is Ϸ95 Å (18.6 bp͞turn), corresponding to that of an active, or ATP-form of the RecA filament. The other is one that has the S-type sugar pucker. Its helical pitch is Ϸ64 Å (12.5 bp͞turn), corresponding to that of an inactive, or ADP-form of the RecA filament. During this modeling, we found that the interconversion of sugar puckers between the N-type and the S-type rotates bases horizontally, while maintaining the deoxyribose-base stacking interaction. We propose that this base rotation enables base pair switching between double-stranded DNA and singlestranded DNA to take place, facilitating homologous pairing and strand exchange. A possible mechanism for strand exchange involving DNA rotation also is discussed.

show abstract

“…In the RecA-mediated pairing and strand exchange reaction, the single-stranded DNA within the nucleoprotein filament binds in the minor groove of the extended Watson-Crick duplex (43)(44)(45). One proposed model for recognition of homology and formation of a nascent joint involves local opening of the base pairs of the duplex, followed by Watson-Crick pairing of the incoming single strand with its complementary strand.…”

Section: Discussionmentioning

confidence: 99%

The Role of Negative Superhelicity and Length of Homology in the Formation of Paranemic Joints Promoted by RecA Protein

Wong¹,

Chiu²,

Chow³

1998

Journal of Biological Chemistry

View full text Add to dashboard Cite

Escherichia coli RecA protein pairs homologous DNA molecules to form paranemic joints when there is an absence of a free end in the region of homologous contact. Paranemic joints are a key intermediate in homologous recombination and are important in understanding the mechanism for a search of homology. The efficiency of paranemic joint formation depended on the length of homology and the topological forms of the duplex DNA. The presence of negative superhelicity increased the pairing efficiency and reduced the minimal length of homology required for paranemic joint formation. Negative superhelicity stimulated joint formation by favoring the initial unwinding of duplex DNA that occurred during the homology search and was not essential in the maintenance of the paired structure. Regardless of length of homology, formation of paranemic joints using circular duplex DNA required the presence of more than six negative supercoils. Above six negative turns, an increasing degree of negative superhelicity resulted in a linear increase in the pairing efficiency. These results support a model of two distinct kinds of DNA unwinding occurring in paranemic joint formation: an initial unwinding caused by heterologous contacts during synapsis and a later one during pairing of the homologous molecules.Escherichia coli RecA protein is a prototype of a class of recombination proteins that are widespread among prokaryotes and eukaryotes (for reviews, see Refs. 1-4). RecA protein is essential in homologous genetic recombination. In vitro, RecA protein polymerizes on single-stranded DNA to form a helical nucleoprotein filament that mediates homologous pairing with duplex DNA and subsequent strand exchange between the two parental molecules. Much of the information on RecA-promoted homologous pairing is derived from a model reaction involving circular single-stranded DNA and linear duplex DNA. In such a system wherein a free DNA end is available, the incoming single strand and its complement in the duplex DNA are free to rotate around each other. The heteroduplex DNA produced has the classical intertwined or plectonemic structure of duplex DNA (5). Since a free end only represents a small fraction of the entire molecule, the apposition of two DNA molecules, especially in vivo, is likely to occur within the interior region at a distance from the ends. RecA protein also promotes the homologous alignment of two DNA substrates in the absence of a free end. The resulting joint is termed paranemic: the DNA strands from the two parental molecules are paired in the homologous region but not topologically linked, and the joint is unstable in the absence of RecA protein (5, 6). Experimentally, paranemic joints can be studied free from plectonemic joints by pairing circular single-stranded DNA with either superhelical DNA or linear "buried homology" duplex DNA in which the homologous region is flanked at both sides by stretches of heterologous sequences (5, 7). Neither combination has net intertwining of the aligned DNA strands nor complete disp...

show abstract

DNA Strand Exchange Mediated by the Escherichia coli RecA Protein Initiates in the Minor Groove of Double-Stranded DNA

Cited by 42 publications

References 40 publications

Construction and evaluation of a kinetic scheme for RecA‐mediated DNA strand exchange

Construction and evaluation of a kinetic scheme for RecA‐mediated DNA strand exchange

Base pair switching by interconversion of sugar puckers in DNA extended by proteins of RecA-family: A model for homology search in homologous genetic recombination

The Role of Negative Superhelicity and Length of Homology in the Formation of Paranemic Joints Promoted by RecA Protein

Contact Info

Product

Resources

About