Intermediates in homologous pairing promoted by recA protein

Tsang, Siu Sing; Muniyappa, K.; Azhderian, E. M.; Gonda, David K.; Radding, Charles M.; Flory, J; Chase, John W.

doi:10.1016/0022-2836(85)90405-x

Cited by 115 publications

(69 citation statements)

References 38 publications

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“…Chymotrypsin was purchased from Sigma-Aldrich. E. coli RecA protein (23), SSB (24), M. tuberculosis RecA (20), and M. smegmatis RecA 2 were purified, and their concentrations were determined as described (25). RecA proteins were purified as described (23).…”

Section: Methodsmentioning

confidence: 99%

Characterization of Single-stranded DNA-binding Proteins from Mycobacteria

Reddy

Guhan

Muniyappa

2001

Journal of Biological Chemistry

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Single-stranded DNA-binding proteins (SSB) play an important role in most aspects of DNA metabolism including DNA replication, repair, and recombination. We report here the identification and characterization of SSB proteins of Mycobacterium smegmatis and Mycobacterium tuberculosis. Sequence comparison of M. smegmatis SSB revealed that it is homologous to M. tuberculosis SSB, except for a small spacer connecting the larger amino-terminal domain with the extreme carboxyl-terminal tail. The purified SSB proteins of mycobacteria bound single-stranded DNA with high affinity, and the association and dissociation constants were similar to that of the prototype SSB. The proteolytic signatures of free and bound forms of SSB proteins disclosed that DNA binding was associated with structural changes at the carboxyl-terminal domain. Significantly, SSB proteins from mycobacteria displayed high affinity for cognate RecA, whereas Escherichia coli SSB did not under comparable experimental conditions. Accordingly, SSB and RecA were coimmunoprecipitated from cell lysates, further supporting an interaction between these proteins in vivo. The carboxyl-terminal domain of M. smegmatis SSB, which is not essential for interaction with ssDNA, is the site of binding of its cognate RecA. These studies provide the first evidence for stable association of eubacterial SSB proteins with their cognate RecA, suggesting that these two proteins might function together during DNA repair and/or recombination.

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Section: Methodsmentioning

confidence: 99%

Characterization of Single-stranded DNA-binding Proteins from Mycobacteria

Reddy

Guhan

Muniyappa

2001

Journal of Biological Chemistry

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“…In homologous pairing, the RecA polypeptide first polymerizes on singlestranded DNA (29,(44)(45)(46) to form a spiral filamentous structure around the DNA (47)(48)(49). The secondary structure of the single-stranded DNA is removed during this step (48), which is stimulated by SSB (single-strand binding protein; refs.…”

Section: Heteroduplex Joint Formation Is Promoted By the Reca͞rad51-fmentioning

confidence: 99%

Homologous genetic recombination as an intrinsic dynamic property of a DNA structure induced by RecA/Rad51-family proteins: A possible advantage of DNA over RNA as genomic material

Shibata

Nishinaka

Mikawa

et al. 2001

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

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Heteroduplex joints are general intermediates of homologous genetic recombination in DNA genomes. A heteroduplex joint is formed between a single-stranded region (or tail), derived from a cleaved parental double-stranded DNA, and homologous regions in another parental double-stranded DNA, in a reaction mediated by the RecA͞Rad51-family of proteins. In this reaction, a RecA͞ Rad51-family protein first forms a filamentous complex with the single-stranded DNA, and then interacts with the double-stranded DNA in a search for homology. Studies of the three-dimensional structures of single-stranded DNA bound either to Escherichia coli RecA or Saccharomyces cerevisiae Rad51 have revealed a novel extended DNA structure. This structure contains a hydrophobic interaction between the 2 methylene moiety of each deoxyribose and the aromatic ring of the following base, which allows bases to rotate horizontally through the interconversion of sugar puckers. This base rotation explains the mechanism of the homology search and base-pair switch between double-stranded and singlestranded DNA during the formation of heteroduplex joints. The pivotal role of the 2 methylene-base interaction in the heteroduplex joint formation is supported by comparing the recombination of RNA genomes with that of DNA genomes. Some simple organisms with DNA genomes induce homologous recombination when they encounter conditions that are unfavorable for their survival. The extended DNA structure confers a dynamic property on the otherwise chemically and genetically stable double-stranded DNA, enabling gene segment rearrangements without disturbing the coding frame (i.e., protein-segment shuffling). These properties may give an extensive evolutionary advantage to DNA.base-pair switch ͉ homologous pairing ͉ strand exchange ͉ NMR ͉ three-dimensional molecular structure DNA as a General Molecular Carrier of Genetic Information G enomic information generally is carried by double-stranded DNA. The double-stranded DNA structure discovered by Watson and Crick clearly explains the mechanisms of heredity, which include both the encoding of genetic information and its duplication as chemical properties of the molecule (1). In addition, the double-stranded structure enables cellular systems to recognize, as structural irregularities, erroneously incorporated nucleotides or lesions in bases, sugars, or the backbone strand, and to correct these errors by using the partner strand as a template. The genetic stability and the chemical inactivity of double-stranded DNA have been regarded as favorable molecular properties for its role as the carrier of genomic information. Evolution, which is a general attribute of the genome as well, has resulted in a variety of organisms, whose diversity arose not only as a result of changes in the genomic information, but also as a result of increased content and complexity. The faithful duplication and repair exhibited by the double-stranded DNA structure would seem to be incompatible with the process of evolution. Thus, evolution has b...

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“…Although these proteins form rings as well as right-handed helical filaments (12)(13)(14), genetic (15), electronmicroscopic (16), and biochemical evidence (17) indicates strongly that the filament promotes homologous pairing and strand exchange. The role or significance of the rings is unknown.…”

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confidence: 99%

The synaptic activity of HsDmc1, a human recombination protein specific to meiosis

Gupta

Golub

et al. 2001

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

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Human Dmc1 protein, a meiosis-specific homolog of Escherichia coli RecA protein, has previously been shown to promote DNA homologous pairing and strand-exchange reactions that are qualitatively similar to those of RecA protein and Rad51. Human and yeast Rad51 proteins each form a nucleoprotein filament that is very similar to the filament formed by RecA protein. However, recent studies failed to find a similar filament made by Dmc1 but showed instead that this protein forms octameric rings and stacks of rings. These observations stimulated further efforts to elucidate the mechanism by which Dmc1 promotes the recognition of homology. Dmc1, purified to a state in which nuclease and helicase activities were undetectable, promoted homologous pairing and strand exchange as measured by fluorescence resonance energy transfer (FRET). Observations on the intermediates and products, which can be distinguished by FRET assays, provided direct evidence of a three-stranded synaptic intermediate. The effects of helix stability and mismatched base pairs on the recognition of homology revealed further that human Dmc1, like human Rad51, requires the preferential breathing of A⅐T base pairs for recognition of homology. We conclude that Dmc1, like human Rad51 and E. coli RecA protein, promotes homologous pairing and strand exchange by a ''synaptic pathway'' involving a three-stranded nucleoprotein intermediate, rather than by a ''helicase pathway'' involving the separation and reannealing of DNA strands.T wo kinds of macromolecular protein structures play prominent roles in homologous genetic recombination: toroids and filaments. Among the toroidal structures, the best understanding of the relation of function to structure exists for Escherichia coli RuvB protein, which forms a hexameric ATPase͞helicase. Two such hexameric rings, interacting with RuvA protein, drive the migration of Holliday structures, the 4-fold branched DNA intermediate formed after initial synapsis and processing have occurred. The consequence of this driven migration is the reciprocal exchange of a pair of like strands between two duplex molecules (1). Rings are also formed by the exonuclease of phage (2), the Erf protein of phage P22 (3), and human Rad52 protein (4, 5). On the basis of the crystal structure of toroidal exonuclease, Kovall and Matthews suggested a mechanistic explanation of the high processivity of this enzyme (2).Some recombination proteins form both rings and filaments: the ␤ protein of phage forms large rings and left-handed helical filaments (6). ␤ protein promotes annealing of complementary single strands (7,8) and migration of a single-stranded branch (9). This protein, which does not hydrolyze ATP, binds more strongly to the product of its annealing activity, which may help to explain how it drives branch migration (10), but there are no correlations of structure with function that help us to understand the role of either the rings or the left-handed filaments. RecT protein, the product of a cryptic prophage in E. coli and a functional ...

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Intermediates in homologous pairing promoted by recA protein

Cited by 115 publications

References 38 publications

Characterization of Single-stranded DNA-binding Proteins from Mycobacteria

Characterization of Single-stranded DNA-binding Proteins from Mycobacteria

Homologous genetic recombination as an intrinsic dynamic property of a DNA structure induced by RecA/Rad51-family proteins: A possible advantage of DNA over RNA as genomic material

The synaptic activity of HsDmc1, a human recombination protein specific to meiosis

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