Deinococcus radiodurans single-stranded DNA binding protein.

摘要

Deinococcus radiodurans RI is one of the most radiation-resistant organisms known and is able to repair an unusually large amount of DNA damage, including DNA double-strand breaks, without induced mutation. Single-stranded DNA-binding proteins (SSBs) are essential proteins in all organisms and are involved in DNA replication, recombination and repair. We show that Deinococcus radiodurans R1 (ATCC 13939) single-stranded DNA binding protein (DrSSB) is encoded by a continuous open reading frame. The biologically active form of DrSSB is homodimeric.; We report the solution of the DrSSB X-ray crystal structure to 1.8-A-resolution. The structure confirms sequence predictions that the protein contains two oligonucleotide/oligosaccharide-binding (OB) domains. These domains are linked by a novel beta-hairpin motif.; We show that DrSSB stimulates both D. radiodurans RecA and Escherichia coli RecA-promoted DNA strand exchange reactions in vitro. We have also shown in vitro that DrSSB promotes a localized melting of the DNA duplex adjacent to single-stranded DNA overhangs as short as 15 nucleotides, with a pronounced duplex destabilization bias for duplexes with a free 3' end when the extension is less than 45 nucleotides. We show that the DrSSB concentration in the cell (∼3000 dimers/cell) is more than sufficient to consider our duplex destabilizing results biologically applicable.; We hypothesize that DrSSB, and perhaps other bacterial SSBs, may locally melt duplexes at frayed ends created by DNA double-strand breaks and act as a signal and/or anchor for other DNA metabolizing proteins. We suggest that SSBs may also act in a like manner at other DNA metabolic intermediates with similar single-strand/double-strand structures.; In a separate investigation, we show that wild-type E. coli can rapidly evolve to be almost as highly radiation resistant as D. radiodurans by using ionizing radiation as selective pressure. Relatively few mutations are necessary to achieve radiation resistance, and these mutations are found in a wide variety of cellular metabolic pathways. The E. coli ssb gene is unchanged in the E. coli radiation resistant strain. We conclude that divergent evolution can lead to extreme microbial radiation resistance.