Combining structural and coevolution information to unveil allosteric sites.

Understanding allosteric regulation in biomolecules is of great interest to pharmaceutical research and computational methods emerged during the last decades to characterize allosteric coupling. However, the prediction of allosteric sites in a protein structure remains a challenging task. Here, we integrate local binding site information, coevolutionary information, and information on dynamic allostery into a structure-based three-parameter model to identify potentially hidden allosteric sites in ensembles of protein structures with orthosteric ligands. When tested on five allosteric proteins (LFA-1, p38-α, GR, MAT2A, and BCKDK), the model successfully ranked all known allosteric pockets in the top three positions. Finally, we identified a novel druggable site in MAT2A confirmed by X-ray crystallography and SPR and a hitherto unknown druggable allosteric site in BCKDK validated by biochemical and X-ray crystallography analyses. Our model can be applied in drug discovery to identify allosteric pockets.

Replisome-mediated translesion synthesis by a cellular replicase.

Genome integrity relies on the ability of the replisome to navigate ubiquitous DNA damage during DNA replication. The Escherichia coli replisome transiently stalls at leading-strand template lesions and can either reinitiate replication downstream of the lesion or recruit specialized DNA polymerases that can bypass the lesion via translesion synthesis. Previous results had suggested that the E. coli replicase might play a role in lesion bypass, but this possibility has not been tested in reconstituted DNA replication systems. We report here that the DNA polymerase III holoenzyme in a stalled E. coli replisome can directly bypass a single cyclobutane pyrimidine dimer or abasic site by translesion synthesis in the absence of specialized translesion synthesis polymerases. Bypass efficiency was proportional to deoxynucleotide concentrations equivalent to those found in vivo and was dependent on the frequency of primer synthesis downstream of the lesion. Translesion synthesis came at the expense of lesion-skipping replication restart. Replication of a cyclobutane pyrimidine dimer was accurate, whereas replication of an abasic site resulted in mainly -1 frameshifts. Lesion bypass was accompanied by an increase in base substitution frequency for the base preceding the lesion. These findings suggest that DNA damage at the replication fork can be replicated directly by the replisome without the need to activate error-prone pathways.

Diabetic kidney disease patients on hemodialysis: a retrospective survival analysis across different socioeconomic groups.

BACKGROUND: Diabetic kidney disease is the leading cause of stage 5 chronic kidney disease (CKD) in India. Renal replacement therapy (RRT) is accessible to very few patients because of socioeconomic deprivation. We studied the effect of diabetes and socioeconomic status on the outcome of patients on maintenance hemodialysis (MHD). METHODS: We retrospectively analyzed the outcome of 897 patients (629 males/268 females; mean age ± standard deviation 48.69 ± 14.27 years) initiated on MHD from 2003 to 2009 at five dialysis centers in south India. There were 335 type 2 diabetic patients and 562 non-diabetic patients. Group 1 comprised the self-paying patients (518 patients) and Group 2 included the TANKER Foundation charity dialysis patients (379 patients). We compared the 5-year survival rates of Group 1 versus Group 2 and also those of diabetic versus non-diabetic patients, using the Kaplan-Meier survival estimator. RESULTS: Of the 897 patients, 166 patients survived, 350 died, 234 were lost to follow-up, 137 had renal transplantation and 10 patients were transferred to peritoneal dialysis. The 5-year survival rates after censoring were 20.7 and 38.2% for diabetic and non-diabetic patients, respectively (P < 0.001). The survival rate of diabetic patients was significantly lower, compared with non-diabetic patients, in Group 2 (P < 0.001), but not significantly lower in Group 1 (P = 0.226). CONCLUSIONS: Diabetic patients have poor survival rates on MHD, especially those from poor socioeconomic groups. Due to scarce RRT facilities and poor survival rates of diabetic patients, prevention, early detection and management of diabetic CKD patients should be the way to go forward.

Visualizing the Nonhomogeneous Structure of RAD51 Filaments Using Nanofluidic Channels.

RAD51 is the key component of the homologous recombination pathway in eukaryotic cells and performs its task by forming filaments on DNA. In this study we investigate the physical properties of RAD51 filaments formed on DNA using nanofluidic channels and fluorescence microscopy. Contrary to the bacterial ortholog RecA, RAD51 forms inhomogeneous filaments on long DNA in vitro, consisting of several protein patches. We demonstrate that a permanent "kink" in the filament is formed where two patches meet if the stretch of naked DNA between the patches is short. The kinks are readily seen in the present microscopy approach but would be hard to identify using conventional single DNA molecule techniques where the DNA is more stretched. We also demonstrate that protein patches separated by longer stretches of bare DNA roll up on each other and this is visualized as transiently overlapping filaments. RAD51 filaments can be formed at several different conditions, varying the cation (Mg(2+) or Ca(2+)), the DNA substrate (single-stranded or double-stranded), and the RAD51 concentration during filament nucleation, and we compare the properties of the different filaments formed. The results provide important information regarding the physical properties of RAD51 filaments but also demonstrate that nanofluidic channels are perfectly suited to study protein-DNA complexes.

Noncognate DNA damage prevents the formation of the active conformation of the Y-family DNA polymerases DinB and DNA polymerase κ.

Y-family DNA polymerases are specialized to copy damaged DNA, and are associated with increased mutagenesis, owing to their low fidelity. It is believed that the mechanism of nucleotide selection by Y-family DNA polymerases involves conformational changes preceding nucleotidyl transfer, but there is limited experimental evidence for such structural changes. In particular, nucleotide-induced conformational changes in bacterial or eukaryotic Y-family DNA polymerases have, to date, not been extensively characterized. Using hydrogen-deuterium exchange mass spectrometry, we demonstrate here that the Escherichia coli Y-family DNA polymerase DinB and its human ortholog DNA polymerase κ undergo a conserved nucleotide-induced conformational change in the presence of undamaged DNA and the correct incoming nucleotide. Notably, this holds true for damaged DNA containing N(2) -furfuryl-deoxyguanosine, which is efficiently copied by these two polymerases, but not for damaged DNA containing the major groove modification O(6) -methyl-deoxyguanosine, which is a poor substrate. Our observations suggest that DinB and DNA polymerase κ utilize a common mechanism for nucleotide selection involving a conserved prechemical conformational transition promoted by the correct nucleotide and only preferred DNA substrates.

Steric gate residues of Y-family DNA polymerases DinB and pol kappa are crucial for dNTP-induced conformational change.

Discrimination against ribonucleotides by DNA polymerases is critical to preserve DNA integrity. For many DNA polymerases, including those of the Y family, rNTP discrimination has been attributed to steric clashes between a residue near the active site, the steric gate, and the 2'-hydroxyl of the incoming rNTP. Here we used hydrogen/deuterium exchange (HDX) mass spectrometry (MS) to probe the effects of the steric gate in the Y-family DNA polymerases Escherichia coli DinB and human DNA pol κ. Formation of a ternary complex with a G:dCTP base pair in the active site resulted in slower hydrogen exchange relative to a ternary complex with G:rCTP in the active site. The protection from exchange was localized to regions both distal and proximal to the active site, suggesting that DinB and DNA pol κ adopt different conformations depending on the sugar of the incoming nucleotide. In contrast, when the respective steric gate residues were mutated to alanine, the differences in HDX between the dNTP- and rNTP-bound ternary complexes were attenuated such that for DinB(F13A) and pol κ(Y112A), ternary complexes with either G:dCTP or G:rCTP base pairs had similar HDX profiles. Furthermore, the HDX in these ternary complexes resembled that of the rCTP-bound state rather than the dCTP-bound state of the wild-type enzymes. Primer extension assays confirmed that DinB(F13A) and pol κ(Y112A) do not discriminate against rNTPs to the same extent as the wild-type enzymes. Our observations indicate that the steric gate is crucial for rNTP discrimination because of its role in specifically promoting a dNTP-induced conformational change and that rNTP discrimination occurs in a relatively closed state of the polymerases.

Conformational analysis of processivity clamps in solution demonstrates that tertiary structure does not correlate with protein dynamics.

The relationship between protein sequence, structure, and dynamics has been elusive. Here, we report a comprehensive analysis using an in-solution experimental approach to study how the conservation of tertiary structure correlates with protein dynamics. Hydrogen exchange measurements of eight processivity clamp proteins from different species revealed that, despite highly similar three-dimensional structures, clamp proteins display a wide range of dynamic behavior. Differences were apparent both for structurally similar domains within proteins and for corresponding domains of different proteins. Several of the clamps contained regions that underwent local unfolding with different half-lives. We also observed a conserved pattern of alternating dynamics of the α helices lining the inner pore of the clamps as well as a correlation between dynamics and the number of salt bridges in these α helices. Our observations reveal that tertiary structure and dynamics are not directly correlated and that primary structure plays an important role in dynamics.

Polymerase manager protein UmuD directly regulates Escherichia coli DNA polymerase III α binding to ssDNA.

Replication by Escherichia coli DNA polymerase III is disrupted on encountering DNA damage. Consequently, specialized Y-family DNA polymerases are used to bypass DNA damage. The protein UmuD is extensively involved in modulating cellular responses to DNA damage and may play a role in DNA polymerase exchange for damage tolerance. In the absence of DNA, UmuD interacts with the α subunit of DNA polymerase III at two distinct binding sites, one of which is adjacent to the single-stranded DNA-binding site of α. Here, we use single molecule DNA stretching experiments to demonstrate that UmuD specifically inhibits binding of α to ssDNA. We predict using molecular modeling that UmuD residues D91 and G92 are involved in this interaction and demonstrate that mutation of these residues disrupts the interaction. Our results suggest that competition between UmuD and ssDNA for α binding is a new mechanism for polymerase exchange.

Selective disruption of the DNA polymerase III α-ß complex by the umuD gene products.

DNA polymerase III (DNA pol III) efficiently replicates the Escherichia coli genome, but it cannot bypass DNA damage. Instead, translesion synthesis (TLS) DNA polymerases are employed to replicate past damaged DNA; however, the exchange of replicative for TLS polymerases is not understood. The umuD gene products, which are up-regulated during the SOS response, were previously shown to bind to the α, ß and ε subunits of DNA pol III. Full-length UmuD inhibits DNA replication and prevents mutagenic TLS, while the cleaved form UmuD' facilitates mutagenesis. We show that α possesses two UmuD binding sites: at the N-terminus (residues 1-280) and the C-terminus (residues 956-975). The C-terminal site favors UmuD over UmuD'. We also find that UmuD, but not UmuD', disrupts the α-ß complex. We propose that the interaction between α and UmuD contributes to the transition between replicative and TLS polymerases by removing α from the ß clamp.