The success of genome-wide association studies has paralleled the development of efficient genotyping technologies. We describe the development of a next-generation microarray based on the new highly-efficient Affymetrix Axiom genotyping technology that we are using to genotype individuals of European ancestry from the Kaiser Permanente Research Program on Genes, Environment and Health (RPGEH). The array contains 674,517 SNPs, and provides excellent genome-wide as well as gene-based and candidate-SNP coverage. Coverage was calculated using an approach based on imputation and cross validation. Preliminary results for the first 80,301 saliva-derived DNA samples from the RPGEH demonstrate very high quality genotypes, with sample success rates above 94% and over 98% of successful samples having SNP call rates exceeding 98%. At steady state, we have produced 462 million genotypes per week for each Axiom system. The new array provides a valuable addition to the repertoire of tools for large scale genome-wide association studies.

Four custom Axiom genotyping arrays were designed for a genome-wide association (GWA) study of 100,000 participants from the Kaiser Permanente Research Program on Genes, Environment and Health. The array optimized for individuals of European race/ethnicity was previously described. Here we detail the development of three additional microarrays optimized for individuals of East Asian, African American, and Latino race/ethnicity. For these arrays, we decreased redundancy of high-performing SNPs to increase SNP capacity. The East Asian array was designed using greedy pairwise SNP selection. However, removing SNPs from the target set based on imputation coverage is more efficient than pairwise tagging. Therefore, we developed a novel hybrid SNP selection method for the African American and Latino arrays utilizing rounds of greedy pairwise SNP selection, followed by removal from the target set of SNPs covered by imputation. The arrays provide excellent genome-wide coverage and are valuable additions for large-scale GWA studies.

Formation of the mature 3 ends of the vast majority of cellular mRNAs occurs through cleavage and polyadenylation and requires a cleavage and polyadenylation specificity factor (CPSF) containing, among other proteins, CPSF-73 and CPSF-100. These two proteins belong to a superfamily of zinc-dependent ␤-lactamase fold proteins with catalytic specificity for a wide range of substrates including nucleic acids. CPSF-73 contains a zinc-binding histidine motif involved in catalysis in other members of the ␤-lactamase superfamily, whereas CPSF-100 has substitutions within the histidine motif and thus is unlikely to be catalytically active. Here we describe two previously unknown human proteins, designated RC-68 and RC-74, which are related to CPSF-73 and CPSF-100 and which form a complex in HeLa and mouse cells. RC-68 contains the intact histidine motif, and hence it might be a functional counterpart of CPSF-73, whereas RC-74 lacks this motif, thus resembling CPSF-100. In HeLa cells RC-68 is present in both the cytoplasm and the nucleus whereas RC-74 is exclusively nuclear. RC-74 does not interact with CPSF-73, and neither RC-68 nor RC-74 is found in a complex with CPSF-160, indicating that these two proteins form a separate entity independent of the CPSF complex and are likely involved in a pre-mRNA processing event other than cleavage and polyadenylation of the vast majority of cellular pre-mRNAs. RNA interference-mediated depletion of RC-68 arrests HeLa cells early in G 1 phase, but surprisingly the arrested cells continue growing and reach the size typical of G 2 cells. RC-68 is highly conserved from plants to humans and may function in conjunction with RC-74 in the 3 end processing of a distinct subset of cellular pre-mRNAs encoding proteins required for G 1 progression and entry into S phase.In metazoans, there are two distinct mechanisms of 3Ј end processing of pre-mRNAs that lead to formation of mature mRNAs. The vast majority of pre-mRNAs are processed at the 3Ј end by a coupled cleavage and polyadenylation reaction (9,57,71,78). However, the replication-dependent histone premRNAs are processed by a one-step mechanism that involves only a cleavage reaction (13,40,47). Both mechanisms require two cis-acting elements in the pre-mRNA and involve different sets of trans-acting factors.In mammalian pre-mRNAs, the cleavage and polyadenylation reaction occurs about 20 nucleotides downstream of a highly conserved polyadenylation signal, AAUAAA. The AA UAAA sequence is recognized by cleavage and polyadenylation specificity factor 160 (CPSF-160) (32, 49), which exists in a stable complex with three other proteins, CPSF-100, CPSF-73, and CPSF-30 (2, 4, 31, 33, 48). The CPSF complex also contains a fifth component, Fip1, which may be only loosely associated with the other subunits or which may exist in nonequimolar amounts in the complex (34). The CPSF complex is required for both cleavage and polyadenylation. About 30 nucleotides downstream of the cleavage site there is a weakly conserved GU-rich element, which is recogni...

The 3 end of mammalian histone mRNAs consisting of a conserved stem-loop and a terminal ACCCA interacts with a recently identified human 3 exonuclease designated 3hExo. The sequence-specific interaction suggests that 3hExo may participate in the degradation of histone mRNAs. ERI-1, a Caenorhabditis elegans homologue of 3hExo, has been implicated in degradation of small interfering RNAs. We introduced a number of mutations to 3hExo to identify residues required for RNA binding and catalysis. To assure that the introduced mutations specifically target one of these two activities of 3hExo rather than cause global structural defects, the mutant proteins were tested in parallel for the ability both to bind the stem-loop RNA and to degrade RNA substrates. Our analysis confirms that 3hExo is a member of the DEDDh family of 3 exonucleases. Specific binding to the RNA requires the SAP domain and two lysines located immediately to its C terminus. 3hExo binds with the highest affinity to the wild-type 3 end of histone mRNA, and any changes to this sequence reduce efficiency of binding. 3hExo has only residual, if any, 3 exonuclease activity on DNA substrates and localizes mostly to the cytoplasm, suggesting that in vivo it performs exclusively RNA-specific functions. Efficient degradation of RNA substrates by 3hExo requires 2 and 3 hydroxyl groups at the last nucleotide. 3hExo removes 3 overhangs of small interfering RNAs, whereas the double-stranded region is resistant to the enzymatic activity.Mammalian histone mRNAs end with a highly conserved and unique stem-loop structure followed by a single-stranded ACCCA sequence (1). The 3Ј end of histone mRNAs is specifically recognized by two proteins, the stem-loop-binding protein (SLBP) and 3ЈhExo (2). Binding of SLBP requires the nucleotides at the 5Ј side of the stem-loop, whereas binding of 3ЈhExo requires the single-stranded ACCCA at the 3Ј end. In addition, binding of each protein requires specific nucleotides in the stem and the loop. SLBP and 3ЈhExo can bind the 3Ј end of histone mRNA either individually or simultaneously, forming a ternary complex (2). SLBP stimulates binding of 3ЈhExo to the stem-loop and allows 3ЈhExo to form a complex with suboptimal RNA targets, raising the possibility that the two proteins make direct contact with each other in the ternary complex (2). Binding of SLBP to the stem-loop in histone mRNA precursors is required for formation of the correct 3Ј end of histone mRNAs in the nucleus (3). SLBP bound to the stem-loop accompanies mature histone mRNA to the cytoplasm, where it stimulates histone mRNA translation (4).The half-life of histone mRNAs is greatly reduced in response to completion or inhibition of DNA replication, resulting in rapid disappearance of histone mRNAs from the cytoplasm and cessation of histone production (5). The stemloop structure is both necessary and sufficient for the selective degradation of histone mRNA and confers the same type of regulation on other mRNAs when introduced at their 3Ј end (6). Sequence-specific and ...

Molecular dynamics (MD) simulations of HhaI DNA methyltransferase and statistical coupling analysis (SCA) data on the DNA cytosine methyltransferase family were combined to identify residues that are coupled by coevolution and motion. The highest ranking correlated pairs from the data matrix product (SCA⅐MD) are colocalized and form stabilizing interactions; the anticorrelated pairs are separated on average by 30 Å and form a clear focal point centered near the active site. We suggest that these distal anticorrelated pairs are involved in mediating active-site compressions that may be important for catalysis. Mutants that disrupt the implicated interactions support the validity of our combined SCA⅐MD approach.anticorrelated motion ͉ correlated motion ͉ M.HhaI ͉ statistical coupling analysis T he proposal that protein dynamics contributes significantly to enzyme catalysis is intriguing (1-4) yet is supported by limited experimental evidence. Previous studies have shown that correlated and anticorrelated motions within an enzyme's active site enhance the reaction rate by various mechanisms that increase the relative amounts of reactive orientations (5). These active-site fluctuations are proposed to result from motions involving distal structural elements and interconnecting networks (1-4). This hypothesis is indirectly supported by emerging molecular dynamics (MD) (1, 2), NMR (6, 7), and hybrid approaches (8-12). The MD studies, although difficult to verify experimentally, have provided highly suggestive results relating dynamics to catalysis. Ultimately, the quantitative contribution to catalysis of various dynamic mechanisms requires direct experimental testing. We combined MD simulations and a coevolution analysis [statistical coupling analysis (SCA); ref. 13] to identify residues that are coupled by coevolution and motion.Although MD simulations reveal active-site correlated and anticorrelated motions, the identity and role of specific structural elements outside the active site in mediating such motions is difficult to assign. For example, MD cross-correlation analyses are dominated by anticorrelated motions occurring between the most distal regions of protein, often residing in distinct domains (5). Although MD simulations implicate regions of allowed motion, the identity of single amino acids that facilitate these motions is not forthcoming and hence difficult for protein engineers to test. SCA identifies the functional coupling of specific residue pairs that in many cases are distal in the three-dimensional structure. The coupling of such residues leads to their coevolution and is revealed by the statistical analysis of hundreds of related sequences; this approach recently was validated by NMR and protein engineering studies (13-16). These applications of SCA have been focused on protein-ligand interactions, and here we apply SCA toward protein dynamics and catalysis.M.HhaI is one of many S-adenosylmethionine (AdoMet)-dependent DNA-modifying enzymes found in bacteria, plants, and animals (17). These enzym...

Cytochrome P450cam (CYP101) is a prokaryotic monooxygenase that requires two proteins, putidaredoxin reductase (PdR) and putidaredoxin (Pdx), to supply electrons from NADH. This study addresses the mechanism by which electrons are transported from PdR to P450cam through Pdx and used to activate O(2) at the heme of P450cam. It is shown that k(cat)/Km(O2) is independent of the PdR concentration and hyperbolically dependent on Pdx. The phenomenon of saturation of reaction rates with either P450cam or PdR at high ratios of one enzyme to the other is investigated and shown to be consistent with a change in the rate limiting step. Either the reduction of Pdx by PdR (high P450) or the reduction of P450 by Pdx (high PdR) determines the rate. These data support a mechanism where Pdx acts as a shuttle for transport of electrons from PdR to P450cam, effectively ruling out the formation of a kinetically significant PdR/Pdx/P450cam complex.

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The early steps in dioxygen activation by the monooxygenase cytochrome P450cam (CYP101) include binding of O2 to ferrous P450cam to yield the ferric-superoxo form (oxyP450cam) followed by an irreversible, long-range electron transfer from putidaredoxin to reduce the oxyP450cam. The steady state kinetic parameter kcat/Km(O2) has been studied by a variety of probes that indicate a small D2O solvent isotope effect (1.21 +/- 0.08), a very small solvent viscosogen effect, and a 16O/18O isotope effect of 1.0147 +/- 0.0007. This latter value, which can be compared with the 16O/18O equilibrium isotope effect of 1.0048 +/- 0.0003 measured for oxyP450cam formation, is attributed to a primarily rate-limiting outer-sphere electron transfer from the heme iron center as O2 that has prebound to protein approaches the active site cofactor. The electron transfer from putidaredoxin to oxyP450cam was investigated by rapid mixing at 25 degrees C to complement previous lower-temperature measurements. A rate of 390 +/- 23 s-1 (and a near-unity solvent isotope effect) supports the view that the long-range electron transfer from reduced putidaredoxin to oxyP450cam is rapid relative to dissociation of O2 from the enzyme. P450cam represents the first enzymatic reaction of O2 in which both equilibrium and kinetic 16O/18O isotope effects have been measured.