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Biochemical characterization of a mutant RecA protein altered in DNA-binding loop 1.

Mirshad, Jk Stephen Charles Kowalczykowski

Published in Biochemistry

The double substitution of Glu156 with Leu and Gly157 with Val in the Escherichia coli RecA protein results in a severely reduced level of recombination and constitutive coprotease behavior. Here we present our examination of the biochemical properties of this mutant protein, RecA N99, in an effort to understand its phenotype and the role of loop 1...

Biochemical basis of the constitutive coprotease activity of RecA P67W protein.

Mirshad, Jk Stephen Charles Kowalczykowski

Published in Biochemistry

The mutation of Pro67 to Trp (P67W) in the Escherichia coli RecA protein results in reduced recombination and constitutive coprotease phenotypes. We examined the biochemical properties of this mutant in an effort to understand these altered behaviors. We find that RecA P67W protein can access single-stranded DNA (ssDNA) binding sites within regions...

Cooperative and noncooperative binding of protein ligands to nucleic acid lattices: experimental approaches to the deter...

Stephen Charles Kowalczykowski Paul, Ls Lonberg, N Newport, Jw Mcswiggen, Ja Von Hippel, Ph

Published in Biochemistry

Many biologically important proteins bind nonspecifically, and often cooperatively, to single-or double-stranded nucleic acid lattices in discharging their physiological functions. This binding can generally be described in thermodynamic terms by three parameters: n, the binding site size; K, the intrinsic binding constant; omega, the binding coope...

Translocation of E. coli RecQ helicase on single-stranded DNA.

Rad, B Stephen Charles Kowalczykowski

Published in Biochemistry

A member of the SF2 family of helicases, Escherichia coli RecQ, is involved in the recombination and repair of double-stranded DNA breaks and single-stranded DNA (ssDNA) gaps. Although the unwinding activity of this helicase has been studied biochemically, the mechanism of translocation remains unclear. To this end, using ssDNA of varying lengths, ...

Enhancement of Escherichia coli RecA protein enzymatic function by dATP.

Menetski, Jp Stephen Charles Kowalczykowski

Published in Biochemistry

The Escherichia coli recA protein has been shown to hydrolyze several nucleoside triphosphates in the presence of ssDNA. The substitution of dATP for rATP has significant effects on various recA protein biochemical properties. In the presence of dATP, recA protein can invade more secondary structure in native ssDNA than it can in the presence of rA...

Atomic force microscope measurements of nucleosome cores assembled along defined DNA sequences.

Allen, Mj Dong, Xf O Neill, Te Yau, P Stephen Charles Kowalczykowski Gatewood, J Balhorn, R Bradbury, Em

Published in Biochemistry

We have found that the atomic force microscope (AFM) can be used to image the "beads-on-a-string" chromatin structure in a normal air environment following adsorption onto a cover glass substrate. Individual nucleosome cores and linker DNA could be resolved clearly along chromatin fibers that were reconstituted using histone octamers and a tandemly...

Enzymatic properties of the RecA803 protein, a partial suppressor of recF mutations.

Madiraju, Mv Lavery, Pe Stephen Charles Kowalczykowski Clark, Aj

Published in Biochemistry

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 D...

Characterization of the adenosinetriphosphatase activity of the Escherichia coli RecBCD enzyme: relationship of ATP hydr...

Roman, Lj Stephen Charles Kowalczykowski

Published in Biochemistry

We find that the rate of dsDNA-dependent ATPase activity is biphasic, with a fast component which represents the unwinding of the dsDNA and a slow component which results from the ssDNA-dependent ATPase activity of recBCD enzyme. Comparison of the ATPase and helicase activities permits evaluation of the efficiency of ATP hydrolysis during unwinding...

Properties of the high-affinity single-stranded DNA binding state of the Escherichia coli recA protein.

Menetski, Jp Varghese, A Stephen Charles Kowalczykowski

Published in Biochemistry

The properties of the high-affinity single-stranded DNA (ssDNA) binding state of Escherichia coli recA protein have been studied. We find that all of the nucleoside triphosphates that are hydrolyzed by recA protein induce a high-affinity ssDNA binding state. The effect of ATP binding to recA protein was partially separated from the ATP hydrolytic e...

A single-stranded DNA binding protein from Drosophila melanogaster: characterization of the heterotrimeric protein and i...

Mitsis, Pg Stephen Charles Kowalczykowski Lehman, Ir

Published in Biochemistry

We describe the purification to near homogeneity of a single-stranded DNA binding protein from 0-18-h embryos of Drosophila melanogaster. Drosophila SSB (D-SSB) is a heterotrimer with subunits of molecular weight of 70,000, 30,000, and 8000. It has a Stokes radius of 48.6 +/- 2 A and s20,w = 5.0 +/- 0.2 S. The interaction of D-SSB with ssDNA was ex...

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