No Sliding during Homology Search by RecA Protein

Adzuma, Kenji

doi:10.1074/jbc.273.47.31565

Cited by 47 publications

(44 citation statements)

References 49 publications

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“…Such dsDNA translocating activity has never been reported for the RecA/ hRad51 family of proteins. Correspondingly, it was shown that the search for homology promoted by RecA protein involves collision rather than extensive sliding of dsDNA (55). Our results indicate that hRad54 protein may drastically change the mechanism of homology search by promoting ATPase-dependent translocation of dsDNA along the filament.…”

Section: Human Rad54p Stimulates Hrad51p In the Presence Of Ca 2ϩmentioning

confidence: 72%

Human Rad54 Protein Stimulates DNA Strand Exchange Activity of hRad51 Protein in the Presence of Ca2+

Mazina

Mazin

2004

Journal of Biological Chemistry

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Rad51 and Rad54 proteins play a key role in homologous recombination in eukaryotes. Recently, we reported that Ca 2؉ is required in vitro for human Rad51 protein to form an active nucleoprotein filament that is important for the search of homologous DNA and for DNA strand exchange, two critical steps of homologous recombination. Here we find that Ca 2؉ is also required for hRad54 protein to effectively stimulate DNA strand exchange activity of hRad51 protein. This finding identifies Ca 2؉ as a universal cofactor of DNA strand exchange promoted by mammalian homologous recombination proteins in vitro. We further investigated the hRad54-dependent stimulation of DNA strand exchange. The mechanism of stimulation appeared to include specific interaction of hRad54 protein with the hRad51 nucleoprotein filament. Our results show that hRad54 protein significantly stimulates homology-independent coaggregation of dsDNA with the filament, which represents an essential step of the search for homologous DNA. The results obtained indicate that hRad54 protein serves as a dsDNA gateway for the hRad51-ssDNA filament, promoting binding and an ATP hydrolysisdependent translocation of dsDNA during the search for homologous sequences.

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Section: Human Rad54p Stimulates Hrad51p In the Presence Of Ca 2ϩmentioning

confidence: 72%

Human Rad54 Protein Stimulates DNA Strand Exchange Activity of hRad51 Protein in the Presence of Ca2+

Mazina

Mazin

2004

Journal of Biological Chemistry

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“…Being completely disordered (27), the in vitro networks cannot, however, alter the dimensionality of the search (30). In contrast, the intracellular assemblies may promote a homology search by both an attenuated sampling volume that results from RecA-DNA coaggregation, and a reduced dimensionality which specifically stems from the longitudinal organization of the in vivo assembly.…”

Section: Discussionmentioning

confidence: 99%

Ordered intracellular RecA–DNA assemblies: A potential site of in vivo RecA-mediated activities

Levin-Zaidman

Frenkiel-Krispin

Shimoni

et al. 2000

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

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The inducible SOS response increases the ability of bacteria to cope with DNA damage through various DNA repair processes in which the RecA protein plays a central role. Here we present the first study of the morphological aspects that accompany the SOS response in Escherichia coli. We find that induction of the SOS system in wild-type bacteria results in a fast and massive intracellular coaggregation of RecA and DNA into a lateral macroscopic assembly. The coaggregates comprise substantial portions of both the cellular RecA and the DNA complement. The structural features of the coaggregates and their relation to in vitro RecA-DNA networks, as well as morphological studies of strains carrying RecA mutants, are all consistent with the possibility that the intracellular assemblies represent a functional entity in which RecA-mediated DNA repair and protection activities occur.biocrystallization ͉ DNA repair ͉ homology search ͉ stress response R ecA-mediated DNA recombination and repair processes proceed through several sequential phases (1-3). A presynaptic filament in which RecA molecules coat a single-stranded DNA substrate is initially formed. The filament acts then as a sequence-specific DNA-binding entity, capable of searching and binding dsDNA sites that are homologous to the RecA-coated segment. Within the resulting joint species, DNA strand exchange and heteroduplex extension processes are promoted. The mechanism that enables a rapid search for DNA homology in vivo, within a highly crowded and complex genome, remains enigmatic.To reach its target, any sequence-specific DNA-binding protein must overcome two general obstacles: a minute cellular concentration of the target, and a vast excess of nontarget, yet still competitive, DNA sites. The search for a homologous DNA site conducted by the RecA-DNA presynaptic filament shares these hurdles, but is further encumbered by the uniquely adverse diffusion characteristics of its components. A DNA target corresponds to a segment that is part of, and embedded within, the chromosome. This, and the large structural asymmetry of DNA, conspire to minimize the diffusion constant of DNA sites (4, 5). In a homology search executed by the RecA-DNA filament, both the searching and the target entities are chromosomal DNA sites whose small diffusion constants drastically attenuate their encounter rate. How then does a RecA-mediated intracellular search evade the kinetic impediments that are intrinsic to the nature of its components?Here we show that damages inflicted on bacterial DNA lead to a rapid formation of an ordered intracellular assembly that accommodates both RecA and DNA. We suggest that the striated morphology of this RecA-DNA assembly is capable of promoting an in vivo homology search by attenuating both the sampling volume and the dimensionality of the process. Moreover, RecA was shown to protect chromosomal DNA from degradation (6) through unknown mechanisms. The tight crystalline packaging that is progressively assumed by the intracellular RecA-DNA assemblies as ...

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“…Single-molecule fluorescent measurements with a high spatiotemporal resolution were employed, which, in contrast to earlier experiments that indicated no sliding (35), have observed multiple diffusional events for RecA filaments on DNA. Using computer simulations, it was argued that sliding accelerates the homology search as much as 200 times (13).…”

Section: Introductionmentioning

confidence: 95%

On the Mechanism of Homology Search by RecA Protein Filaments

Kochugaeva

Shvets

Kolomeisky

2017

Biophysical Journal

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Genetic stability is a key factor in maintaining, survival and reproduction of biological cells. It relies on many processes, but one of the most important is a homologous recombination, in which the repair of breaks in double-stranded DNA molecules is taking place with a help of several specific proteins. In bacteria this task is accomplished by RecA proteins that are active as nucleoprotein filaments formed on single-stranded segments of DNA. A critical step in the homologous recombination is a search for a corresponding homologous region on DNA, which is called a homology search. Recent single-molecule experiments clarified some aspects of this process, but its molecular mechanisms remain not well understood. We developed a quantitative theoretical approach to analyze the homology search. It is based on a discrete-state stochastic model that takes into account the most relevant physical-chemical processes in the system. Using a method of first-passage processes, a full dynamic description of the homology search is presented. It is found that the search dynamics depends on the degree of extension of DNA molecules and on the size of RecA nucleoprotein filaments, in agreement with experimental single-molecule measurements of DNA pairing by RecA proteins. Our theoretical calculations, supported by extensive Monte Carlo computer simulations, provide a molecular description of the mechanisms of the homology search.

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No Sliding during Homology Search by RecA Protein

Cited by 47 publications

References 49 publications

Human Rad54 Protein Stimulates DNA Strand Exchange Activity of hRad51 Protein in the Presence of Ca2+

Human Rad54 Protein Stimulates DNA Strand Exchange Activity of hRad51 Protein in the Presence of Ca2+

Ordered intracellular RecA–DNA assemblies: A potential site of in vivo RecA-mediated activities

On the Mechanism of Homology Search by RecA Protein Filaments

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