scholarly journals Characterization of an ATP-dependent DNA strand transferase from human cells.

1987 ◽  
Vol 7 (9) ◽  
pp. 3124-3130 ◽  
Author(s):  
D Ganea ◽  
P Moore ◽  
L Chekuri ◽  
R Kucherlapati
Keyword(s):  
Dna Sequences ◽  
Atp Hydrolysis ◽  
Reca Protein ◽  
Strand Exchange ◽  
Cell Nuclei ◽  
In Vitro Recombination ◽  
Dna Strand ◽  
Exchange Activity

We have characterized an enzymatic activity from human cell nuclei which is capable of catalyzing strand exchange between homologous DNA sequences. The strand exchange activity was Mg2+ dependent and required ATP hydrolysis. In addition, it was capable of promoting reannealing of homologous DNA sequences and could form nucleoprotein networks in a fashion reminiscent of purified bacterial RecA protein. Using an in vitro recombination assay, we also showed that the strand exchange activity was biologically important. The factor(s) responsible for the activity has been partially purified.

Characterization of an ATP-dependent DNA strand transferase from human cells

1987 ◽  
Vol 7 (9) ◽  
pp. 3124-3130
Author(s):  
D Ganea ◽  
P Moore ◽  
L Chekuri ◽  
R Kucherlapati
Keyword(s):  
Dna Sequences ◽  
Atp Hydrolysis ◽  
Reca Protein ◽  
Strand Exchange ◽  
Cell Nuclei ◽  
In Vitro Recombination ◽  
Dna Strand ◽  
Exchange Activity

We have characterized an enzymatic activity from human cell nuclei which is capable of catalyzing strand exchange between homologous DNA sequences. The strand exchange activity was Mg2+ dependent and required ATP hydrolysis. In addition, it was capable of promoting reannealing of homologous DNA sequences and could form nucleoprotein networks in a fashion reminiscent of purified bacterial RecA protein. Using an in vitro recombination assay, we also showed that the strand exchange activity was biologically important. The factor(s) responsible for the activity has been partially purified.

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

2004 ◽  
Vol 279 (50) ◽  
pp. 52042-52051 ◽  
Author(s):  
Olga M. Mazina ◽  
Alexander V. Mazin
Keyword(s):  
Homologous Recombination ◽  
Atp Hydrolysis ◽  
Specific Interaction ◽  
Strand Exchange ◽  
Rad51 Protein ◽  
Recombination Proteins ◽  
Dna Strand Exchange ◽  
Dna Strand ◽  
Exchange Activity

Rad51 and Rad54 proteins play a key role in homologous recombination in eukaryotes. Recently, we reported that Ca2+is requiredin vitrofor 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 Ca2+is also required for hRad54 protein to effectively stimulate DNA strand exchange activity of hRad51 protein. This finding identifies Ca2+as a universal cofactor of DNA strand exchange promoted by mammalian homologous recombination proteinsin 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 hydrolysis-dependent translocation of dsDNA during the search for homologous sequences.

scholarly journals DNA Helicase Activity of PcrA Is Not Required for the Displacement of RecA Protein from DNA or Inhibition of RecA-Mediated Strand Exchange

2007 ◽  
Vol 189 (12) ◽  
pp. 4502-4509 ◽  
Author(s):  
Syam P. Anand ◽  
Haocheng Zheng ◽  
Piero R. Bianco ◽  
Sanford H. Leuba ◽  
Saleem A. Khan
Keyword(s):  
Dna Recombination ◽  
Reca Protein ◽  
Resonance Energy ◽  
Dna Helicase ◽  
Strand Exchange ◽  
Single Pair ◽  
Helicase Activity ◽  
Dna Strand Exchange ◽  
Dna Strand

ABSTRACT PcrA is a conserved DNA helicase present in all gram-positive bacteria. Bacteria lacking PcrA show high levels of recombination. Lethality induced by PcrA depletion can be overcome by suppressor mutations in the recombination genes recFOR. RecFOR proteins load RecA onto single-stranded DNA during recombination. Here we test whether an essential function of PcrA is to interfere with RecA-mediated DNA recombination in vitro. We demonstrate that PcrA can inhibit the RecA-mediated DNA strand exchange reaction in vitro. Furthermore, PcrA displaced RecA from RecA nucleoprotein filaments. Interestingly, helicase mutants of PcrA also displaced RecA from DNA and inhibited RecA-mediated DNA strand exchange. Employing a novel single-pair fluorescence resonance energy transfer-based assay, we demonstrate a lengthening of double-stranded DNA upon polymerization of RecA and show that PcrA and its helicase mutants can reverse this process. Our results show that the displacement of RecA from DNA by PcrA is not dependent on its translocase activity. Further, our results show that the helicase activity of PcrA, although not essential, might play a facilitatory role in the RecA displacement reaction.

scholarly journals RecA Protein from the Extremely Radioresistant Bacterium Deinococcus radiodurans: Expression, Purification, and Characterization

2002 ◽  
Vol 184 (6) ◽  
pp. 1649-1660 ◽  
Author(s):  
Jong-Il Kim ◽  
Ajay K. Sharma ◽  
Stephen N. Abbott ◽  
Elizabeth A. Wood ◽  
David W. Dwyer ◽  
...  
Keyword(s):  
Deinococcus Radiodurans ◽  
Atp Hydrolysis ◽  
Reca Protein ◽  
Strand Exchange ◽  
Exchange Reactions ◽  
Ssdna Binding ◽  
E Coli ◽  
Dna Strand Exchange ◽  
Ssdna Binding Protein ◽  
Dna Strand

ABSTRACT The RecA protein of Deinococcus radiodurans (RecADr) is essential for the extreme radiation resistance of this organism. The RecADr protein has been cloned and expressed in Escherichia coli and purified from this host. In some respects, the RecADr protein and the E. coli RecA (RecAEc) proteins are close functional homologues. RecADr forms filaments on single-stranded DNA (ssDNA) that are similar to those formed by the RecAEc. The RecADr protein hydrolyzes ATP and dATP and promotes DNA strand exchange reactions. DNA strand exchange is greatly facilitated by the E. coli SSB protein. As is the case with the E. coli RecA protein, the use of dATP as a cofactor permits more facile displacement of bound SSB protein from ssDNA. However, there are important differences as well. The RecADr protein promotes ATP- and dATP-dependent reactions with distinctly different pH profiles. Although dATP is hydrolyzed at approximately the same rate at pHs 7.5 and 8.1, dATP supports an efficient DNA strand exchange only at pH 8.1. At both pHs, ATP supports efficient DNA strand exchange through heterologous insertions but dATP does not. Thus, dATP enhances the binding of RecADr protein to ssDNA and the displacement of ssDNA binding protein, but the hydrolysis of dATP is poorly coupled to DNA strand exchange. The RecADr protein thus may offer new insights into the role of ATP hydrolysis in the DNA strand exchange reactions promoted by the bacterial RecA proteins. In addition, the RecADr protein binds much better to duplex DNA than the RecAEc protein, binding preferentially to double-stranded DNA (dsDNA) even when ssDNA is present in the solutions. This may be of significance in the pathways for dsDNA break repair in Deinococcus.

Repair of deletions and double-strand gaps by homologous recombination in a mammalian in vitro system

1991 ◽  
Vol 11 (1) ◽  
pp. 445-457
Author(s):  
R Jessberger ◽  
P Berg
Keyword(s):  
Dna Sequences ◽  
Kanamycin Resistance ◽  
Double Strand Breaks ◽  
Strand Transfer ◽  
In Vitro System ◽  
Strand Breaks ◽  
Nuclear Extracts ◽  
Dna Strand

We have designed an in vitro system using mammalian nuclear extracts, or fractions derived from them, that can restore the sequences missing at double-strand breaks (gaps) or in deletions. The recombination substrates consist of (i) recipient DNA, pSV2neo with gaps or deletions ranging from 70 to 390 bp in the neo sequence, and (ii) donor DNAs with either complete homology to the recipient (pSV2neo) or plasmids whose homology with pSV2neo is limited to a 1.0- to 1.3-kbp neo segment spanning the gaps or deletions. Incubation of these substrates with various enzyme fractions results in repair of the recipient DNA's disrupted neo gene. The recombinational repair was monitored by transforming recA Escherichia coli to kanamycin resistance and by a new assay which measures the extent of DNA strand transfer from the donor substrate to the recipient DNA. Thus, either streptavidin- or antidigoxigenin-tagged beads are used to separate the biotinylated or digoxigeninylated recipient DNA, respectively, after incubation with the isotopically labeled donor DNA. In contrast to the transfection assay, the DNA strand transfer measurements are direct, quantitative, rapid, and easy, and they provide starting material for the characterization of the recombination products and intermediates. Accordingly, DNA bound to beads serves as a suitable template for the polymerase chain reaction. With appropriate pairs of oligonucleotide primers, we have confirmed that both gaps and deletions are fully repaired, that deletions can be transferred from the recipient DNA to the donor's intact neo sequence, and that cointegrant molecules containing donor and recipient DNA sequences are formed.

scholarly journals Repair of deletions and double-strand gaps by homologous recombination in a mammalian in vitro system.

1991 ◽  
Vol 11 (1) ◽  
pp. 445-457 ◽  
Author(s):  
R Jessberger ◽  
P Berg
Keyword(s):  
Dna Sequences ◽  
Kanamycin Resistance ◽  
Double Strand Breaks ◽  
Strand Transfer ◽  
In Vitro System ◽  
Strand Breaks ◽  
Nuclear Extracts ◽  
Dna Strand

We have designed an in vitro system using mammalian nuclear extracts, or fractions derived from them, that can restore the sequences missing at double-strand breaks (gaps) or in deletions. The recombination substrates consist of (i) recipient DNA, pSV2neo with gaps or deletions ranging from 70 to 390 bp in the neo sequence, and (ii) donor DNAs with either complete homology to the recipient (pSV2neo) or plasmids whose homology with pSV2neo is limited to a 1.0- to 1.3-kbp neo segment spanning the gaps or deletions. Incubation of these substrates with various enzyme fractions results in repair of the recipient DNA's disrupted neo gene. The recombinational repair was monitored by transforming recA Escherichia coli to kanamycin resistance and by a new assay which measures the extent of DNA strand transfer from the donor substrate to the recipient DNA. Thus, either streptavidin- or antidigoxigenin-tagged beads are used to separate the biotinylated or digoxigeninylated recipient DNA, respectively, after incubation with the isotopically labeled donor DNA. In contrast to the transfection assay, the DNA strand transfer measurements are direct, quantitative, rapid, and easy, and they provide starting material for the characterization of the recombination products and intermediates. Accordingly, DNA bound to beads serves as a suitable template for the polymerase chain reaction. With appropriate pairs of oligonucleotide primers, we have confirmed that both gaps and deletions are fully repaired, that deletions can be transferred from the recipient DNA to the donor's intact neo sequence, and that cointegrant molecules containing donor and recipient DNA sequences are formed.

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