Complex formation by the human Rad51B and Rad51C DNA repair proteins and their activities in vitro

Yi Ching Lio, Alexander V. Mazin, Stephen C. Kowalczykowski, David J. Chen

Research output: Contribution to journalArticle

101 Citations (Scopus)

Abstract

The human Rad51 protein is essential for DNA repair by homologous recombination. In addition to Rad51 protein, five paralogs have been identified: Rad51B/Rad51L1, Rad51C/Rad51L2, Rad51D/Rad51L3, XRCC2, and XRCC3. To further characterize a subset of these proteins, recombinant Rad51, Rad51B-(His)6, and Rad51C proteins were individually expressed employing the baculovirus system, and each was purified from Sf9 insect cells. Evidence from nickel-nitrilotriacetic acid pull-down experiments demonstrates a highly stable Rad51B·Rad51C heterodimer, which interacts weakly with Rad51. Rad51B and Rad51C proteins were found to bind single- and double-stranded DNA and to preferentially bind 3′-end-tailed double-stranded DNA. The ability to bind DNA was elevated with mixed Rad51 and Rad51C, as well as with mixed Rad51B and Rad51C, compared with that of the individual protein. In addition, both Rad51B and Rad51C exhibit DNA-stimulated ATPase activity. Rad51C displays an ATP-independent apparent DNA strand exchange activity, whereas Rad51B shows no such activity; this apparent strand exchange ability results actually from a duplex DNA destabilization capability of Rad51C. By analogy to the yeast Rad55 and Rad57, our results suggest that Rad51B and Rad51C function through interactions with the human Rad51 recombinase and play a crucial role in the homologous recombinational repair pathway.

Original languageEnglish (US)
Pages (from-to)2469-2478
Number of pages10
JournalJournal of Biological Chemistry
Volume278
Issue number4
DOIs
StatePublished - Jan 24 2003

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DNA Repair
Repair
DNA
Recombinational DNA Repair
Proteins
His-His-His-His-His-His
Rad51 Recombinase
Sf9 Cells
Baculoviridae
Single-Stranded DNA
Recombinant Proteins
Insects
Adenosine Triphosphatases
In Vitro Techniques
Yeast
Adenosine Triphosphate
Yeasts
Experiments

ASJC Scopus subject areas

  • Biochemistry

Cite this

Complex formation by the human Rad51B and Rad51C DNA repair proteins and their activities in vitro. / Lio, Yi Ching; Mazin, Alexander V.; Kowalczykowski, Stephen C.; Chen, David J.

In: Journal of Biological Chemistry, Vol. 278, No. 4, 24.01.2003, p. 2469-2478.

Research output: Contribution to journalArticle

Lio, Yi Ching ; Mazin, Alexander V. ; Kowalczykowski, Stephen C. ; Chen, David J. / Complex formation by the human Rad51B and Rad51C DNA repair proteins and their activities in vitro. In: Journal of Biological Chemistry. 2003 ; Vol. 278, No. 4. pp. 2469-2478.
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abstract = "The human Rad51 protein is essential for DNA repair by homologous recombination. In addition to Rad51 protein, five paralogs have been identified: Rad51B/Rad51L1, Rad51C/Rad51L2, Rad51D/Rad51L3, XRCC2, and XRCC3. To further characterize a subset of these proteins, recombinant Rad51, Rad51B-(His)6, and Rad51C proteins were individually expressed employing the baculovirus system, and each was purified from Sf9 insect cells. Evidence from nickel-nitrilotriacetic acid pull-down experiments demonstrates a highly stable Rad51B·Rad51C heterodimer, which interacts weakly with Rad51. Rad51B and Rad51C proteins were found to bind single- and double-stranded DNA and to preferentially bind 3′-end-tailed double-stranded DNA. The ability to bind DNA was elevated with mixed Rad51 and Rad51C, as well as with mixed Rad51B and Rad51C, compared with that of the individual protein. In addition, both Rad51B and Rad51C exhibit DNA-stimulated ATPase activity. Rad51C displays an ATP-independent apparent DNA strand exchange activity, whereas Rad51B shows no such activity; this apparent strand exchange ability results actually from a duplex DNA destabilization capability of Rad51C. By analogy to the yeast Rad55 and Rad57, our results suggest that Rad51B and Rad51C function through interactions with the human Rad51 recombinase and play a crucial role in the homologous recombinational repair pathway.",
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