Complete nucleotide sequence of the<i>Escherichia coli recB</i>gene

Finch, Paul W.; Storey, Alan; Chapman, Karen; Brown, Katharine A Robson; Hickson, Ian D.; Emmerson, Peter T.

doi:10.1093/nar/14.21.8573

Cited by 79 publications

(38 citation statements)

References 48 publications

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“…We have also detected a homology at the amino acid level to nucleotide-binding sequences that are present in a number of proteins binding and hydrolyzing ATP, such as the RAD 18 and RAD3 proteins from yeast (Jones et al 1988) and the Escherichia coli proteins UvrA (Hussain et al 1986), UvrD (Finch and Emmerson 1984), RecA (Walker et al 1982), and RecB (Finch et al 1986). Many of these proteins are involved in DNA repair, helicase activity, or recombination.…”

Section: Discussionmentioning

confidence: 88%

xlgv7: a maternal gene product localized in nuclei of the central nervous system in Xenopus laevis.

Miller¹,

Kloc²,

Reddy³

et al. 1989

Genes Dev.

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The Xenopus oocyte nucleus (GV) is a storehouse for a large number of proteins that are used during early development. We have cloned and characterized a cDNA coding for a maternal gene product that is localized in the GV and then becomes highly enriched in the nuclei of the central nervous system {CNS) of tadpoles and adult frogs. This cDNA {xlgv7) is 2.1 kb and hybridizes to a 2.4-kb RNA species on Northern blots. Southern blots of genomic DNA suggest that this gene is a member of a multigene family. The cDNA sequence reveals a long open reading frame (ORF) of 1773 nucleotides, with a putative nuclear targeting signal {Glu Arg Arg Lys Lys Lys Thr) at the extreme carboxyl terminus and an internal histidine {His)-rich region with a repeated conserved amino acid sequence between His pairs. The significance of this region is unclear, but the protein is a DNA-binding protein, and it is possible that this region is involved in this function. The xlgv7 protein also possesses a putative nucleotide-binding consensus sequence that is similar to the bacterial RecA and RecB and yeast RAD proteins. Protein xlgv7 exists as several isotypes that exhibit developmental and cellspecific changes during development. Northern blot analysis of the abundance of the xlgv7 mRNA shows an accumulation following neural induction at stages 15--16. There is a transient expression of the mRNA in the gut of tadpoles. In the adult, the mRNA is highly enriched in the brain and is absent or in very low abundance in other tissues. Immunohistochemical analysis of the protein shows that the protein is localized in the nuclei of the brain cells. We conclude that the xlgv7 gene product is a maternal protein that may serve several important functions, one of which may be in the development and maintanance of the CNS.

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Section: Discussionmentioning

confidence: 88%

xlgv7: a maternal gene product localized in nuclei of the central nervous system in Xenopus laevis.

Miller¹,

Kloc²,

Reddy³

et al. 1989

Genes Dev.

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“…The correlation between intercistronic length and the efficiency of reinitiation in eucaryotes is curiously just the opposite of what occurs in procaryotes. In many, albeit not all (2,47), coregulated bacterial genes, the terminator codon of one cistron overlaps the initiator codon of the next (6,9,15,23,43,49,53,72,73) and translation of the downstream cistron is drastically impaired when the intercistronic distance is expanded experimentally (Fig. 6).…”

Section: Discussionmentioning

confidence: 99%

Effects of intercistronic length on the efficiency of reinitiation by eucaryotic ribosomes.

Kozak¹

1987

Mol. Cell. Biol.

502

433

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Simian virus 40-based plasmids that direct the synthesis of preproinsulin during short-term transfection of COS cells have been used to probe the mechanism of reinitiation by eucaryotic ribosomes. Earlier studies from several laboratories had established that the ability of ribosomes to reinitiate translation at an internal AUG codon depends on having a terminator codon in frame with the preceding AUG triplet and upstream from the intended restart site. In the present studies, the position of the upstream terminator codon relative to the preproinsulin restart site has been systematically varied. The efficiency of reinitiation progressively improved as the intercistronic sequence was lengthened. When the upstream "minicistron" terminated 79 nucleotides before the preproinsulin start site, the synthesis of proinsulin was as efficient as if there were no upstream AUG codons. A mechanism is postulated that might account for this result, which is somewhat surprising inasmuch as bacterial ribosomes reinitiate less efficiently as the intercistronic gap is widened.Eucaryotic ribosomes usually initiate at the AUG codon that lies closest to the 5' end of the mRNA (31). That tendency has been rationalized by postulating that the small ribosomal subunit binds initially at the capped 5' end of the mRNA and subsequently migrates to the AUG initiator codon, which is recognized more or less efficiently depending on nearby sequences. From a comparison of several hundred vertebrate mRNAs, GCCACCAUGG has been proposed as the consensus sequence for initiation in higher eucaryotes (30, 33, Kozak, submitted for publication). The contribution of every nucleotide in that motif has been confirmed by mutagenesis (36; Kozak, J. Mol. Biol., in press). The most highly conserved nucleotides are the purine, most often A, in position -3 (i.e., three nucleotides upstream from the AUG codon) and the G in position +4; the aforementioned mutational analyses confirmed that those two positions have the strongest influence on initiation. Thus, a potential initiator codon can usually be designated as "strong" or "weak" by considering only those positions.There are ways for eucaryotic ribosomes to initiate at internal sites in certain mRNAs, but those sites can be reached only by advancing from the 5' end; never, it seems, by direct internal binding. An early indication of the constraint against direct internal binding was the inability of eucaryotic ribosomes to bind to circular RNA templates (27,28), and a considerable body of other evidence has since been adduced (see Discussion). One mechanism by which ribosomes can reach an internal AUG codon is "leaky scanning," which occurs when the 5'-proximal AUG codon lies in an unfavorable context for initiation. Data consistent with leaky scanning have come from manipulating the sequences of synthetic constructs (35,36,40) and from analyzing naturally occurring viral mRNAs that are bifunctional (reviewed in references 38 and 39). A second mechanism for reaching an internal AUG codon operates when the upstre...

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“…The specific activity was 325,000 ds exonuclease units per mg of protein, which was measured by A280 and the molar extinction coefficient calculated for RecBCD (5). A conversion factor of 5.6 x 109 enzyme molecules per ds exonuclease unit was used, calculated from the specific activity noted above, from the enzyme's subunit molecular weights deduced from the DNA sequence (25)(26)(27) T4 DNA ligase and ATP to the mixture resulted both in religation of the (now 32P-labeled) Sty I site and in ligation of the Taylomeres onto the EcoRI sites at the ends of the DNA. Inactivation of the DNA ligase, followed by restriction with Nde I, produced two self-complementary DNA molecules, one 2300 base pairs (bp) and 32P-labeled, the other 2000 bp and unlabeled.…”

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

RecBCD enzyme is altered upon cutting DNA at a chi recombination hotspot.

Taylor

Smith

1992

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

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During its unidirectional unwinding of DNA, RecBCD enzyme cuts one DNA strand near a properly oriented Chi site, a hotspot of homologous genetic recombination in Escherichia cohl. We report here that individual DNA molecules containing two properly oriented Chi sites were cut with about 40% efficiency at one or the other Chi site but not detectably at both Chi sites. Furthermore, initial incubation of RecBCD with Chi-containing DNA reduced its ability both to unwind DNA and to cut at Chi sites on subsequently added DNA molecules much more than did initial incubation with Chi-free DNA; the nuclease activity was less severely affected. These results imply that RecBCD loses its Chi-cutting activity upon cutting at a single Chi site and provide a mechanism for ensuring single genetic exchanges near the ends of DNA molecules.RecBCD (EC 3.1.11.5) is a multifunctional enzyme required for homologous recombination by the major (RecBCD) pathway of Escherichia coli (reviewed in refs. 1 and 2). The enzyme unwinds linear duplex DNA, from a flush or nearly flush duplex end (3), with the production of single-stranded (ss) DNA loops that enlarge at about 100 nucleotides (nt) per sec as the enzyme travels along the DNA at about 300 nt per sec (4, 5). The enzyme hydrolyses about two ATP molecules per base pair unwound (6). When the enzyme encounters a Chi site, 5'-GCTGGTGG-3', it frequently nicks the Chicontaining strand about 5 nt to the 3' side of Chi (7,8). Nicking at Chi occurs if the enzyme approaches Chi from the right, as the sequence is written here, but not if it approaches Chi from the left (8). DNA unwinding is postulated to continue after Chi cutting (as shown in Fig. 1), with 3'-ended ss DNA, bearing Chi near its end, being extruded as the enzyme continues to travel along DNA (9). This ss DNA "tail" has been proposed (9) to be a potent substrate for RecA protein, which, together with SSB (ss DNA-binding protein), forms joint molecules between ss DNA and homologous double-stranded (ds) DNA (reviewed in ref. 10). The concomitant action of purified RecBCD enzyme, RecA protein, and SSB produces joint molecules from linear ds DNA and circular, supercoiled ds DNA (11). A Chi site in the linear ds DNA determines the apparent size of these joint molecules: the joint molecules are apparently larger the farther the Chi site is from one end of the linear ds DNA (the end "downstream" or 5' of the Chi site) (12).The RecBCD pathway is the principal pathway of recombination during conjugation and transduction of E. coli. In either case, a linear duplex chromosomal fragment from the donor recombines with a complete circular chromosome in the recipient cell. Consequently, two (or any even number of) exchanges are required to produce a viable (complete, circular) recombinant chromosome. The model in Fig. 1 were grown by chloramphenicol-induced amplification in E. coli N100 (galK recA thyA) and purified by alkaline lysis (20), followed by banding twice in cesium chloride/ethidium bromide equilibrium density gradients. Bacteriop...

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Complete nucleotide sequence of theEscherichia coli recBgene

Abstract: The complete nucleotide sequence of the E §gh2raghi ggli regc gene which encodes a subunit of the ATP-dependent DNase, Exonuclease V, has been determined. The

Cited by 79 publications

References 48 publications

xlgv7: a maternal gene product localized in nuclei of the central nervous system in Xenopus laevis.

xlgv7: a maternal gene product localized in nuclei of the central nervous system in Xenopus laevis.

Effects of intercistronic length on the efficiency of reinitiation by eucaryotic ribosomes.

RecBCD enzyme is altered upon cutting DNA at a chi recombination hotspot.

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