The Need for Open Access Publications in Commercial Clinical Genetic Workflows

Presentation for the 2018 University of North Texas Open Access Symposium. This presentation discusses the adoption (or lack thereof) of Open Access and Open Data trends at for profit healthcare organizations from a scientist’s point of vie

Mutator Suppression and Escape from Replication Error–Induced Extinction in Yeast

Cells rely on a network of conserved pathways to govern DNA replication fidelity. Loss of polymerase proofreading or mismatch repair elevates spontaneous mutation and facilitates cellular adaptation. However, double mutants are inviable, suggesting that extreme mutation rates exceed an error threshold. Here we combine alleles that affect DNA polymerase δ (Pol δ) proofreading and mismatch repair to define the maximal error rate in haploid yeast and to characterize genetic suppressors of mutator phenotypes. We show that populations tolerate mutation rates 1,000-fold above wild-type levels but collapse when the rate exceeds 10−3 inactivating mutations per gene per cell division. Variants that escape this error-induced extinction (eex) rapidly emerge from mutator clones. One-third of the escape mutants result from second-site changes in Pol δ that suppress the proofreading-deficient phenotype, while two-thirds are extragenic. The structural locations of the Pol δ changes suggest multiple antimutator mechanisms. Our studies reveal the transient nature of eukaryotic mutators and show that mutator phenotypes are readily suppressed by genetic adaptation. This has implications for the role of mutator phenotypes in cancer

Polar destabilization of DNA duplexes with single-stranded overhangs by the Deinococcus radiodurans SSB protein. Biochemistry 45:14490–14502

ABSTRACT: The Deinococcus radiodurans SSB protein has an occluded site size of 50 ( 2 nucleotides on ssDNA but can form a stable complex with a 26-30-nucleotide oligodeoxynucleotide using a subset of its four ssDNA binding domains. Quantitative estimates of D. radiodurans SSB protein in the D. radiodurans cell indicate approximately 2500-3000 dimers/cell, independent of the level of irradiation. At biologically relevant concentrations, when bound at single-strand-double-strand DNA junctions in vitro, D. radiodurans SSB protein has a limited capacity to displace the shorter strand of the duplex, permitting it to bind to single-strand extensions shorter than 26-30 nucleotides. The capacity to displace the shorter strand of the duplex shows a pronounced bias for extensions with a free 3′ end. The Escherichia coli SSB protein has a similar but somewhat less robust capacity to displace a DNA strand annealed adjacent to a single-strand extension. These activities are likely to be relevant to the action of bacterial SSB proteins in double-strand break repair, acting at the frayed ends created by ionizing radiation. Deinococcus radiodurans is of particular interest in DNA repair investigation as it is one of the most radiation resistant organisms known (1, 2). D. radiodurans is a soil bacterium featuring a D 37 γ irradiation dose of approximately 6000 Gy, making this organism roughly 200 times more resistant to radiation than Escherichia coli (1, 2). DNA damage is not prevented in D. radiodurans. A 6000 Gy dose introduces approximately 300 DNA double-strand (ds) breaks, more than 3000 single-strand (ss) breaks, and more than 1000 sites of base damage per D. radiodurans haploid genome (refs 1 and 2 and references therein). D. radiodurans repairs its genome with a robust DNA damage repair system. As singlestranded DNA-binding (SSB) proteins are essential to DNA replication, recombination, and repair in all known organisms (3), we have pursued the study of D. radiodurans SSB (DrSSB) in hopes of shedding greater light on the DNA damage repair system of D. radiodurans. The DrSSB protein is unusual among bacterial SSB proteins, having two subunits (each with two OB folds) in place of the much more common four subunits (each with one OB fold) (4, 5). Does this unusual structure play a role in the reconstitution of the Deinococcus genome after irradiation? Bacterial SSB proteins were initially called DNA-unwinding proteins, helix-destabilizing proteins, and other related terms (6). However, the primary focus of SSB protein studies in recent years has been on its single-stranded DNA protection and stabilization characteristics, and the emerging interest in cellular protein-SSB protein interactions (6-18). The traditional helix-destabilizing characteristics of SSB proteins are occasionally still tested in the characterization of newly purified SSB proteins in the form of DNA melting temperature depression assays We show in this report that D. radiodurans SSB (DrSSB) facilitates a local denaturation of the DNA helix at the ssdsDNA junction of partially duplex DNAs with overhangs of both 15 and 30 nucleotides. The denaturation occurs with a strong bias favoring 3′ overhangs. We also show that sequential binding of a second DrSSB protein dimer on longer 60-nucleotide overhangs can also destabilize the DNA helix at the ss-dsDNA junction of partial duplexes. We consider these results to be physiologically relevant as care was taken to include both monovalent and divalent cations, known DNA helix stabilizers, in the experimental design. Additionally, quantitation of DrSSB protein in the D. radiodurans cell shows that the concentration of DrSSB protein in the cell is more than sufficient for duplex destabilization at ss-dsDNA junctions. EXPERIMENTAL PROCEDURES Proteins. The D. radiodurans (Dr) and E. coli (Ec) SSB proteins were purified as described previously (5, 21)

Crystal structure of the Deinococcus radiodurans single-stranded DNA-binding protein suggests a mechanism for coping with DNA damage

Single-stranded DNA (ssDNA)-binding (SSB) proteins are uniformly required to bind and protect single-stranded intermediates in DNA metabolic pathways. All bacterial and eukaryotic SSB proteins studied to date oligomerize to assemble four copies of a conserved domain, called an oligonucleotide/oligosaccharide-binding (OB) fold, that cooperate in nonspecific ssDNA binding. The vast majority of bacterial SSB family members function as homotetramers, with each monomer contributing a single OB fold. However, SSB proteins from the Deinococcus-Thermus genera are exceptions to this rule, because they contain two OB folds per monomer. To investigate the structural consequences of this unusual arrangement, we have determined a 1.8-Å-resolution x-ray structure of Deinococcus radiodurans SSB. The structure shows that D. radiodurans SSB comprises two OB domains linked by a β-hairpin motif. The protein assembles a four-OB-fold arrangement by means of symmetric dimerization. In contrast to homotetrameric SSB proteins, asymmetry exists between the two OB folds of D. radiodurans SSB because of sequence differences between the domains. These differences appear to reflect specialized roles that have evolved for each domain. Extensive crystallographic contacts link D. radiodurans SSB dimers in an arrangement that has important implications for higher-order structures of the protein bound to ssDNA. This assembly utilizes the N-terminal OB domain and the β-hairpin structure that is unique to Deinococcus and Thermus species SSB proteins. We hypothesize that differences between D. radiodurans SSB and homotetrameric bacterial SSB proteins may confer a selective advantage to D. radiodurans cells that aids viability in environments that challenge genomic stability