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Protein Sci. 2014 Oct;23(10):1349-58. doi: 10.1002/pro.2519. Epub 2014 Aug 4.

Interaction of double-stranded DNA with polymerized PprA protein from Deinococcus radiodurans.

Author information

1
Molecular Biology Research Division, Quantum Beam Science Center, Japan Atomic Energy Agency, Ibaraki, 319-1195, Japan.

Abstract

Pleiotropic protein promoting DNA repair A (PprA) is a key protein that facilitates the extreme radioresistance of Deinococcus radiodurans. To clarify the role of PprA in the radioresistance mechanism, the interaction between recombinant PprA expressed in Escherichia coli with several double-stranded DNAs (i.e., super coiled, linear, or nicked circular dsDNA) was investigated. In a gel-shift assay, the band shift of supercoiled pUC19 DNA caused by the binding of PprA showed a bimodal distribution, which was promoted by the addition of 1 mM Mg, Ca, or Sr ions. The dissociation constant of the PprA-supercoiled pUC19 DNA complex, calculated from the relative portions of shifted bands, was 0.6 μM with Hill coefficient of 3.3 in the presence of 1 mM Mg acetate. This indicates that at least 281 PprA molecules are required to saturate a supercoiled pUC19 DNA, which is consistent with the number (280) of bound PprA molecules estimated by the UV absorption of the PprA-pUC19 complex purified by gel filtration. This saturation also suggests linear polymerization of PprA along the dsDNA. On the other hand, the bands of linear dsDNA and nicked circular dsDNA that eventually formed PprA complexes did not saturate, but created larger molecular complexes when the PprA concentration was >1.3 μM. This result implies that DNA-bound PprA aids association of the termini of damaged DNAs, which is regulated by the concentration of PprA. These findings are important for the understanding of the mechanism underlying effective DNA repair involving PprA.

KEYWORDS:

DNA binding; DNA repair; gel-shift assay; polymerization; radiation

PMID:
25044036
PMCID:
PMC4286997
DOI:
10.1002/pro.2519
[Indexed for MEDLINE]
Free PMC Article
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