The structure of the E. coli beta clamp polymerase processivity factor has been solved in complex with primed DNA. Interestingly, the clamp directly binds the DNA duplex and also forms a crystal contact with the ssDNA template strand, which binds into the protein-binding pocket of the clamp. We demonstrate that these clamp-DNA interactions function in clamp loading, perhaps by inducing the ring to close around DNA. Clamp binding to template ssDNA may also serve to hold the clamp at a primed site after loading or during switching of multiple factors on the clamp. Remarkably, the DNA is highly tilted as it passes through the beta ring. The pronounced 22 degrees angle of DNA through beta may enable DNA to switch between multiple factors bound to a single clamp simply by alternating from one protomer of the ring to the other.

The papillomavirus E2 proteins regulate the transcription of all papillomavirus genes and are necessary for viral DNA replication. Disruption of the E2 gene is commonly associated with malignancy in cervical carcinoma, indicating that E2 has a role in regulating tumor progression. Although the E2 proteins from all characterized papillomaviruses bind specifically to the same 12-base pair DNA sequence, the cancer-associated human papillomavirus E2 proteins display a unique ability to detect DNA flexibility and intrinsic curvature. To understand the structural basis for this phenomenon, we have determined the crystal structures of the human papillomavirus-18 E2 DNA-binding domain and its complexes with high and low affinity binding sites. The E2 protein is a dimeric ␤-barrel and the E2-DNA interaction is accompanied by a large deformation of the DNA as it conforms to the E2 surface. DNA conformation and E2-DNA contacts are similar in both high and low affinity complexes. The differences in affinity correlate with the flexibility of the DNA sequence. Preferences of E2 proteins from different papillomavirus strains for flexible or prebent DNA targets correlate with the distribution of positive charge on their DNA interaction surfaces, suggesting a role for electrostatic forces in the recognition of DNA deformability.Networks of hydrogen bonds between amino acid side chains and the functional groups of DNA bases are a well documented mechanism of DNA sequence recognition by proteins. Not as well understood is the stereochemical and energetic basis by which protein-DNA binding affinity is modulated when all of these direct components of interaction are conserved. Biologically, such discriminatory abilities are essential when DNAbinding proteins have to select between multiple binding sites present among a vast excess of nonspecific DNA. This is the case in the papillomaviruses, where the primary transcriptional regulatory protein, E2, is confronted by numerous binding sites on the viral genome (Fig. 1a). These sites must be occupied in a defined order such that transcription and viral DNA replication proceed in a regulated fashion (1-7).The papillomaviruses are a large family of double-stranded DNA viruses that vary in host species specificity, tissue tropism, and the clinical outcome of infection (8, 9). The E2 protein has the same functions in the over 100 characterized papillomavirus strains: to activate or repress transcription in a contextdependent fashion and to facilitate the initiation of viral DNA replication via interactions with the viral replication protein E1. Differences in the details of transcription and replication control have been documented between the viral strains that infect different host tissues and between the papillomavirus strains that are associated with different conditions in humans such as warts or cervical cancer (2-7). The E2 proteins from all viral strains have in common the fact that they bind a palindromic DNA sequence ACCgNNNNcGGT, referred to as the E2 binding site (E2BS; 1 lowerc...

DNA polymerases attach to the DNA sliding clamp through a common overlapping binding site. We identify a small-molecule compound that binds the protein-binding site in the Escherichia coli ␤-clamp and differentially affects the activity of DNA polymerases II, III, and IV. To understand the molecular basis of this discrimination, the cocrystal structure of the chemical inhibitor is solved in complex with ␤ and is compared with the structures of Pol II, Pol III, and Pol IV peptides bound to ␤. The analysis reveals that the small molecule localizes in a region of the clamp to which the DNA polymerases attach in different ways. The results suggest that the small molecule may be useful in the future to probe polymerase function with ␤, and that the ␤-clamp may represent an antibiotic target.antibiotic target ͉ rational drug design ͉ fluorescence anisotropy ͉ crystallography T he replication machinery of all cells utilizes a ring-shaped, sliding-clamp protein that encircles DNA and slides along the duplex, thus acting as a mobile tether to hold the chromosomal replicase to DNA for high processivity (1-3). Sliding clamps from the three domains of life are remarkably similar in architecture (4-6). They consist of six domains of similar chain fold. The bacterial ␤-clamp is a homodimer, and each protomer consists of three domains, whereas eukaryotic and archaeal proliferating cell nuclear antigen (PCNA) clamps are homotrimers formed from protomers containing two domains each.Initially, ␤ and PCNA were identified as processivity factors for chromosomal replicases, but sliding clamps are now known to function with diverse DNA polymerases, repair factors, and cell cycle-control proteins (reviewed in ref.

BackgroundIt is estimated that more than two-thirds of the population in sub-Saharan Africa (SSA) must leave their home to collect water, putting them at risk for a variety of negative health outcomes. There is little research, however, quantifying who is most affected by long water collection times.ObjectivesThis study aims to a) describe gender differences in water collection labor among both adults and children (< 15 years of age) in the households (HHs) that report spending more than 30 minutes collecting water, disaggregated by urban and rural residence; and b) estimate the absolute number of adults and children affected by water collection times greater than 30 minutes in 24 SSA countries.MethodsWe analyzed data from the Demographic Health Survey (DHS) and the Multiple Indicator Cluster Survey (MICS) (2005–2012) to describe water collection labor in 24 SSA countries.ResultsAmong households spending more than 30 minutes collecting water, adult females were the primary collectors of water across all 24 countries, ranging from 46% in Liberia (17,412 HHs) to 90% in Cote d’Ivoire (224,808 HHs). Across all countries, female children were more likely to be responsible for water collection than male children (62% vs. 38%, respectively). Six countries had more than 100,000 households (HHs) where children were reported to be responsible for water collection (greater than 30 minutes): Burundi (181,702 HHs), Cameroon (154,453 HHs), Ethiopia (1,321,424 HHs), Mozambique (129,544 HHs), Niger (171,305 HHs), and Nigeria (1,045,647 HHs).ConclusionIn the 24 SSA countries studied, an estimated 3.36 million children and 13.54 million adult females were responsible for water collection in households with collection times greater than 30 minutes. We suggest that accessibility to water, water collection by children, and gender ratios for water collection, especially when collection times are great, should be considered as key indicators for measuring progress in the water, sanitation and hygiene sector.

SUMMARY Human monoclonal antibodies (mAbs) 447-52D and 537-10D, both coded by the VH3 gene and specific for the third variable region (V3) of the HIV-1 gp120, were found to share antigen binding structural elements including an elongated CDR H3 forming main-chain interactions with the N-terminus of the V3 crown. However, water-mediated hydrogen bonds and a unique cation-π sandwich stacking allow 447-52D to be broadly reactive with V3 containing both the GPGR and GPGQ crown motifs, while the deeper binding pocket, and a buried Glu in the binding site of 537-10D limit its reactivity to only V3 containing the GPGR motif. Our results suggest that the design of immunogens for anti-V3 antibodies should avoid the Arg at the V3 crown, as GPGR-containing epitopes appear to select for B cells making antibodies of narrower specificity than V3 that carry Gln at this position.

Gfh1, a transcription factor from Thermus thermophilus, inhibits all catalytic activities of RNA polymerase (RNAP). We characterized the Gfh1 structure, function and possible mechanism of action and regulation. Gfh1 inhibits RNAP by competing with NTPs for coordinating the active site Mg2+ ion. This coordination requires at least two aspartates at the tip of the Gfh1 N‐terminal coiled‐coil domain (NTD). The overall structure of Gfh1 is similar to that of the Escherichia coli transcript cleavage factor GreA, except for the flipped orientation of the C‐terminal domain (CTD). We show that depending on pH, Gfh1‐CTD exists in two alternative orientations. At pH above 7, it assumes an inactive ‘flipped’ orientation seen in the structure, which prevents Gfh1 from binding to RNAP. At lower pH, Gfh1‐CTD switches to an active ‘Gre‐like’ orientation, which enables Gfh1 to bind to and inhibit RNAP.

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The Dachshund proteins are essential components of a regulatory network controlling cell fate determination. They have been implicated in eye, limb, brain, and muscle development. These proteins cannot be assigned to any recognizable structural or functional class based on amino acid sequence analysis. The 1.65 A crystal structure of the most conserved domain of human DACHSHUND is reported here. The protein forms an alpha/beta structure containing a DNA binding motif similar to that found in the winged helix/forkhead subgroup of the helix-turn-helix family. This unexpected finding alters the previously proposed molecular models for the role of Dachshund in the eye determination pathway. Furthermore, it provides a rational framework for future mechanistic analyses of the Dachshund proteins in several developmental contexts.