Identification of the amino acid substitutions in two mutant forms of the recA protein from Escherichia coli: recA441 and recA629.

Knight, Kendall L.; Aoki, Kazumasa; Ujita, E L; McEntee, Kevin

doi:10.1016/s0021-9258(18)90859-8

Cited by 38 publications

(3 citation statements)

References 20 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The recA441 allele also partially suppresses recF mutations, although differently than recA803 (e.g., recA441 suppresses the regulatory defect due to the recF mutation whereas recA803 does not; Thomas & Lloyd, 1983;Volkert et al, 1984). One of the recA441 mutations is at codon 38 [resulting in a change from glutamic acid to lysine (Knight et al, 1984)], which is adjacent to the recA803 mutation at codon 37 [valine to methionine (Madiraju etal., 1988)]. What makes this comparison all the more interesting is that in vitro, the RecA441 protein shows both enhanced DNA strand exchange activity under suboptimal conditions and enhanced ability to compete with SSB protein for ssDNA binding (Lavery & Kowalczykowski, 1988.…”

Section: Discussionmentioning

confidence: 99%

Enzymic properties of the RecA803 protein, a partial suppressor of recF mutations

Madiraju¹,

Lavery²,

Kowalczykowski³

et al. 1992

Biochemistry

View full text Add to dashboard Cite

The RecA803 protein suppresses the recombinational repair defect of recF mutations and displays enhanced joint molecule formation in vitro (Madiraju et al., 1988). To understand the physical basis for these phenomena, the biochemical properties of RecA803 protein were compared with those of the wild-type protein. The RecA803 protein shows greater DNA-dependent ATPase activity than the wild-type protein with either M13 single-stranded (ss) DNA, which contains secondary structure, or double-stranded DNA. This increased activity reflects an enhanced ability of the mutant protein to form active complexes with these DNA molecules rather than an enhanced catalytic turnover activity, because identical kcat values for ATP hydrolysis are obtained when DNA substrates lacking secondary structure are examined. In addition, the ssDNA-dependent ATPase activity of RecA803 protein displays greater resistance to inhibition by SSB (single-stranded DNA binding) protein. These properties of the RecA803 protein are not due to either an increased binding affinity for ssDNA or an increased kinetic lifetime of RecA803 protein-ssDNA complexes, demonstrating that altered protein-DNA stability is not the basis for the enhanced properties of RecA803 protein. However, the nucleation-limited rate of association with ssDNA is more rapid for the RecA803 protein than for wild-type RecA protein. Consequently, we suggest that altered protein-protein interactions may account for the differences between these two proteins. The implications of these results with regard to the partial suppression of recF mutations by recA803 are discussed (Madiraju et al., 1988).

show abstract

Section: Discussionmentioning

confidence: 99%

Enzymic properties of the RecA803 protein, a partial suppressor of recF mutations

Madiraju¹,

Lavery²,

Kowalczykowski³

et al. 1992

Biochemistry

View full text Add to dashboard Cite

show abstract

“…However, peptides Tlt and TI3 were identified by using 0.15 nmol of the HPLC-purified peptide. The numbering of amino acid residues is that of Sancar et al (1980), and the numbering of tryptic peptides is that of Knight et al (1984). The peak designations are those shown in Figure 6.…”

Section: Resultsmentioning

confidence: 99%

“…Yields were estimated by analytical SDS-polyacrylamide gel electrophoresis using known amounts of single-stranded binding (SSB) protein from Escherichia coli as standards. Although the fragments isolated by this procedure were extremely pure on the basis of SDSpolyacrylamide gel analysis and HPLC examination, in several attempts we were unable to dansylate or to sequence the gel-isolated fragments using procedures that we had used successfully for analyzing tryptic fragments isolated using two-dimensional peptide mapping techniques (Knight et al, 1984).…”

Section: Methodsmentioning

confidence: 99%

Evidence for nucleotide-mediated changes in the domain structure of the RecA protein of Escherichia coli

Kobayashi¹,

Knight²,

McEntee³

1987

Biochemistry

View full text Add to dashboard Cite

We have used limited trypsin digestion as a means of investigating changes in the structural properties of recA protein accompanying the binding of different nucleoside triphosphates. The levels of four partial digestion products are greatly increased in digests of recA protein complexed with dTTP, dATP, ATP, or the ATP analogue adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S). These bands (22, 19, and 17.5 kilodaltons) are absent or present at reduced levels in digests of recA protein alone. Unlike these nucleotides, all of which bind tightly to recA protein, nucleotides and analogues that bind poorly produce little or no change in the digestion pattern of recA protein. We have compared the rates of fragment accumulation in the presence of dTTP and show a saturable dependence on nucleotide concentration. Binding of single-stranded DNA to recA protein does not alter the pattern of digestion products compared to protein alone, and the digestion pattern of recA protein-DNA-ATP gamma S ternary complexes is similar to that of uncomplexed enzyme. We have used monoclonal antibody binding, high-performance liquid chromatography separation of peptides, and amino acid composition analyses to localize the regions of recA protein which are altered in their susceptibility to trypsin when nucleoside triphosphates are present. The results of these analyses indicate that the fragments arise from trypsin cutting at two or more sites near the middle of the primary sequence. These cleavage sites are more than 80-110 residues away from the site of photoaffinity labeling by 8-N3ATP (Tyr-264). Our results suggest that, in the presence of certain nucleotides, recA protein is organized into two stable structural domains.

show abstract

Inducibility of the SOS response in a recA730 or recA441 strain is restored by transformation with a new recA allele

1993

View full text Add to dashboard Cite

Escherichia coli RecA protein plays an essential role in both genetic recombination and SOS repair; in vitro RecA needs to bind ATP to promote both activities. Residue 264 is involved in this interaction; we have therefore created two new recA alleles, recA664 (Tyr264-->Glu) and recA665 (Tyr264-->His) bearing mutations at this site. As expected both mutations affected all RecA activities in vivo. Complementation experiments between these new alleles and wild-type recA or recA441 or recA730 alleles, both of which lead to constitutively activated RecA protein, were performed to further investigate the modulatory effects of these mutants on the regulation of SOS repair/recombination pathways. Our results provide further insight into the process of polymerization of RecA protein and its regulatory functions.

show abstract

Identification of the amino acid substitutions in two mutant forms of the recA protein from Escherichia coli: recA441 and recA629.

Cited by 38 publications

References 20 publications

Enzymic properties of the RecA803 protein, a partial suppressor of recF mutations

Enzymic properties of the RecA803 protein, a partial suppressor of recF mutations

Evidence for nucleotide-mediated changes in the domain structure of the RecA protein of Escherichia coli

Inducibility of the SOS response in a recA730 or recA441 strain is restored by transformation with a new recA allele

Contact Info

Product

Resources

About