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

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 lM 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 lM. This result implies that DNAbound 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.

DNA repair in hyperthermophilic and hyperradioresistant microorganisms

“…The polymerization of PprA depends on the concentration of the protein. Whereas PprA binds to dsDNA to form an unsaturated nucleoprotein complex at low concentrations, a larger nucleoprotein complex is formed to facilitate the association of the ends of damaged DNAs at high concentrations [54 ]. Analyses of the cellular dynamics of PprA have indicated the interactions of PprA with topoisomerase (Topo IB) and DNA gyrase, suggesting the significant role of PprA in preserving genome integrity following irradiation [55][56][57].…”

Extended structure of pleiotropic DNA repair‐promoting protein PprA from Deinococcus radiodurans

“…Although mutations on Ala139 and Trp183 residues inside the protein molecule lost the DNA binding ability by disruption of PprA oligomerization, mutations on the molecular surface near Interface 2 without the disruption of oligomerization may lead to investigating effects of interactions between PprA and gyrase to radioresistance. The binding affinity of PprA, however, is similar for linear and supercoiled DNA (33). PprA therefore seems not to assist DNA relaxation with the deformation of DNA but is a DNA guide for gyrase.…”

“…Wild-type PprA exhibits a wide range of oligomeric structures, even without binding to DNA, and the molecular size of the oligomers is estimated to be from 1 3 10 5 to 3 3 10 6 Da (tetramer to 100-mers), depending on the protein concentration (33). Attempts to crystallize wildtype PprA yielded a rod-shaped single crystal (Supplemental Fig.…”

“…PprA is a pleiotropic protein that is believed to be involved in the repair of DSBs and other radiation-induced damage; however, the molecular mechanism underlying this critical function remains unclear. We have previously characterized the interaction of E. coli-expressed recombinant PprA with several double-stranded DNAs (supercoiled, linear, or nicked circular double-stranded DNA) using a gel shift assay (33). The band shift of supercoiled DNA caused by PprA binding exhibits a bimodal distribution.…”

PprA Protein Is Involved in Chromosome Segregation via Its Physical and Functional Interaction with DNA Gyrase in Irradiated Deinococcus radiodurans Bacteria

“…In vitro , PprA preferentially binds double-stranded DNA (dsDNA) carrying strand breaks, inhibits Escherichia coli exonuclease III activity, and stimulates the DNA end-joining reaction catalyzed by ATP-dependent DNA ligases ( 14 ). It has also been shown that PprA polymerizes along supercoiled, nicked, circular, or linear double-stranded DNA ( 23 ). After irradiation, PprA is part of a multiprotein complex containing 24 proteins, including DNA ligases, DNA topoisomerase IB (Topo IB), SSB, and DNA polymerase I and exhibiting both DNA synthesis and DNA end-processing functions ( 24 ).…”