recA mutations that reduce the constitutive coprotease activity of the RecA1202(Prtc) protein: possible involvement of interfilament association in proteolytic and recombination activities

Liu, S. K.; Eisen, Jonathan A.; Hanawalt, Philip C.; Tessman, Irwin

doi:10.1128/jb.175.20.6518-6529.1993

Cited by 25 publications

(22 citation statements)

References 59 publications

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“…Thus, instead of sampling the genome through a three-dimensional random drift, RecA-DNA presynaptic filaments may search for homologous sites by a one-dimensional slide along the dsDNA, whose parallel arrangement coincides with the main axis of the assembly. On the basis of a mutational analysis of RecA (31,32) and its crystal structure (16), it was proposed that RecA-DNA filaments and dsDNA molecules align to form a functional bundle-like coaggregate (32). This model is consistent with the striated morphology of the intracellular assembly (Figs.…”

Section: Discussionsupporting

confidence: 62%

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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Section: Discussionsupporting

confidence: 62%

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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“…Activation of RecA in the cell proceeds from the binding of RecA to ssDNA (62,63), and thus the binding to dsDNA is normally suppressed. That is why the expression of a C-terminal truncated or mutated protein in the cell can lead to constitutive expression of the SOS response (64,65).…”

Section: Discussionmentioning

confidence: 99%

Domain structure and dynamics in the helical filaments formed by RecA and Rad51 on DNA

Xiong

Jacobs

West

et al. 2001

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

228

247

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“…On the other hand, the introduction of a new positive charge by the Q-1843K amino acid change in protein RecA1202 produces enhancement of protein binding to single-stranded DNA that is consistent with such properties of the protein as a higher level of LexA repressor cleavage and its coprotease constitutive activity (24). In turn, the latter can be reduced by a neighboring K-1773Q substitution, generated by an intragenic suppressor recA1630 mutation (13), probably because of the loss of positively charged residue Lys-177. This is the reason why we constructed the substitution K-1833M with the loss of positively charged residue Lys-183.…”

Section: Resultsmentioning

confidence: 96%

“…While it does not participate in an intrafilament interaction, it does take part in an interfilament interaction. The latter has been recently discussed as a possible step in the recombination exchange reaction (13).…”

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confidence: 99%

Genetic characteristics of new recA mutants of Escherichia coli K-12

et al. 1996

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In vivo, the RecA protein of Escherichia coli plays a critical role in such cellular functions as homologous recombination, repair of damaged DNA, and the SOS response (see references 10, 12, and 21 for reviews). To mediate necessary biochemical reactions, the RecA protein must be in an active form that combines three ingredients (RecA, ATP, and singlestranded DNA) in a helical RecA nucleoprotein filament (17).The current view of the physical structure of the filament is based on X-ray analysis of RecA crystals (23). According to that analysis, the RecA monomer is composed of three domains, one major domain and two subdomains located at its amino and carboxyl termini. Both the major domain and the N-terminal domain participate in the formation of RecA multimers, the structure of which has been suggested to resemble closely that of the RecA-DNA filament (23). The C-terminal domain protrudes from the multimer. While it does not participate in an intrafilament interaction, it does take part in an interfilament interaction. The latter has been recently discussed as a possible step in the recombination exchange reaction (13).The C-terminal domain consists of three ␣ helices (H, I, and J) that are situated orthogonally and two ␤ strands (␤9 and ␤10) located between the H and I helices (23). Epitope mapping of anti-RecA protein monoclonal immunoglobulins G, ARM191 and ARM193, which were suggested to affect, respectively, the site for interaction of RecA monomers within the RecA filament and the site for interaction between RecA and double-stranded DNA, showed that the antibodies cover the region of the C-terminal domain including at least ␤-strand 10, as well as helices I and J (7). The functional role of ␣-helix H and adjacent ␤-strand 9 remains unclear. We used sitedirected mutagenesis to elucidate the possible functions of these two adjacent structures.The other reason for our mutagenesis study was to search for functionally thermosensitive (FT) mutations. To date, only two FT mutations, recA44 (8) and recA200 (14), have been described. The former has been localized. We sequenced the recA2283 allele, which was responsible for the RecA200 FT phenotype. The analysis of both recA44 and recA2283 mutations helped us to find a strategy for searching for other thermosensitive mutations in this and other regions. The rationale was to design amino acid substitutions which must be functionally related to the original residues.Besides the C-terminal domain, two regions of the major domain, ␤-strand 6 and ␣-helix F, were involved in the construction of new mutations. The choice of the former region was dictated by an expectation, based on the properties of known recA mutations from this area, that it should be possible to reveal the site responsible for regulation of interaction between the RecA protein and the LexA repressor (see reference 19 for a review). The ␣-helix F region belongs to the type of polypeptide motifs predicted by Churchill and Travers (3) as that which might recognize structural features of DNA.In this repor...

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recA mutations that reduce the constitutive coprotease activity of the RecA1202(Prtc) protein: possible involvement of interfilament association in proteolytic and recombination activities

Cited by 25 publications

References 59 publications

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

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

Domain structure and dynamics in the helical filaments formed by RecA and Rad51 on DNA

Genetic characteristics of new recA mutants of Escherichia coli K-12

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