Changes in the National Cancer Institute's R01 workforce: growth, aging, retention, and policy implications.

Scientific progress and discovery of preventions and cures for life-threatening diseases depend on the vitality of the biomedical research workforce. We analyzed the workforce of cancer researchers applying for and receiving R01 awards from the National Cancer Institute (NCI) from fiscal years 1990 to 2016, the last year prior to implementation of the Next Generation Researchers Initiative. Here we report that the NCI R01 Principal Investigator (PI) workforce expanded 1.4-fold and aged over this time frame. We tracked 9 age groups and found that the number of PIs in the 3 oldest groups increased dramatically, in contrast with the younger groups. Sustained increases in the number of funded older PIs stemmed from increases in the number of older PIs submitting applications, rather than higher funding rates for older PIs. The decline in the number of funded younger PIs was driven in part by (a) a marked increase in time from PhD degree to first R01 application and award, as well as (b) a decrease in retention of PIs in the funded R01 workforce beyond their first R01 award. The NCI is using these and other analyses to inform strategies and policies for attracting, supporting, and retaining meritorious early-career researchers.

Direct measurement of DNA bending by type IIA topoisomerases: implications for non-equilibrium topology simplification.

Type IIA topoisomerases modify DNA topology by passing one segment of duplex DNA (transfer or T-segment) through a transient double-strand break in a second segment of DNA (gate or G-segment) in an ATP-dependent reaction. Type IIA topoisomerases decatenate, unknot and relax supercoiled DNA to levels below equilibrium, resulting in global topology simplification. The mechanism underlying this non-equilibrium topology simplification remains speculative. The bend angle model postulates that non-equilibrium topology simplification scales with the bend angle imposed on the G-segment DNA by the binding of a type IIA topoisomerase. To test this bend angle model, we used atomic force microscopy and single-molecule Förster resonance energy transfer to measure the extent of bending imposed on DNA by three type IIA topoisomerases that span the range of topology simplification activity. We found that Escherichia coli topoisomerase IV, yeast topoisomerase II and human topoisomerase IIα each bend DNA to a similar degree. These data suggest that DNA bending is not the sole determinant of non-equilibrium topology simplification. Rather, they suggest a fundamental and conserved role for DNA bending in the enzymatic cycle of type IIA topoisomerases.

Use of divalent metal ions in the DNA cleavage reaction of topoisomerase IV.

It has long been known that type II topoisomerases require divalent metal ions in order to cleave DNA. Kinetic, mutagenesis and structural studies indicate that the eukaryotic enzymes utilize a novel variant of the canonical two-metal-ion mechanism to promote DNA scission. However, the role of metal ions in the cleavage reaction mediated by bacterial type II enzymes has been controversial. Therefore, to resolve this critical issue, this study characterized the DNA cleavage reaction of Escherichia coli topoisomerase IV. We utilized a series of divalent metal ions with varying thiophilicities in conjunction with oligonucleotides that replaced bridging and non-bridging oxygen atoms at (and near) the scissile bond with sulfur atoms. DNA scission was enhanced when thiophilic metal ions were used with substrates that contained bridging sulfur atoms. In addition, the metal-ion dependence of DNA cleavage was sigmoidal in nature, and rates and levels of DNA cleavage increased when metal ion mixtures were used in reactions. Based on these findings, we propose that topoisomerase IV cleaves DNA using a two-metal-ion mechanism in which one of the metal ions makes a critical interaction with the 3'-bridging atom of the scissile phosphate and facilitates DNA scission by the bacterial type II enzyme.

Aminoglycoside resistance by ArmA-mediated ribosomal 16S methylation in human bacterial pathogens.

Aminoglycosides are a medically important class of antibiotics used to treat serious infections. Methylation of the ribosomal target is an emerging mechanism that produces a high level of resistance to all clinically available aminoglycosides for systemic therapy except streptomycin. ArmA was the first methyltransferase using this mechanism to be discovered in a clinical isolate. We demonstrate that ArmA methylates the N7 position of nucleotide G1405 in 16S rRNA. Methylation at this position is presumed to mediate cellular resistance by blocking aminoglycoside binding by ribosomes. To test this hypothesis, we measured the binding of gentamicin by 30S subunits. Under our conditions, we did not observe binding by ribosomes methylated by ArmA. Furthermore, the ArmA methylation reaction is specific for the 30S ribosomal subunit; neither 16S rRNA alone nor the 70S ribosome is a substrate for this reaction under our experimental conditions, implicating ribosomal proteins in substrate recognition. The biochemical characteristics of ArmA place it in the Agr family of methyltransferases, whose members are predominantly anti-suicide genes from Actinomycetes aminoglycoside producers. The discrepancy between the 30% GC content of armA and the >60% GC content of Actinomycetes, however, calls into question the origin of armA. We demonstrate that surprisingly, the natural promoter of armA from gram-negative Klebsiella pneumoniae was active in gram-positive Bacillus subtilis, suggesting that armA originated from a low-GC, gram-positive aminoglycoside-producing organism.

Building-block selectivity of polyketide synthases.

For the past decade, polyketide synthases have presented an exciting paradigm for the controlled manipulation of complex natural product structure. These multifunctional enzymes catalyze the biosynthesis of polyketide natural products by stepwise condensation and modification of metabolically derived building blocks. In particular, regioselective modification of polyketide structure is possible by alterations in either intracellular acyl-CoA pools or, more commonly, by manipulation of acyl transferases that act as the primary gatekeepers for building blocks.

Quantitative analysis of loading and extender acyltransferases of modular polyketide synthases.

The acyltransferase (AT) domains of modular polyketide synthases (PKSs) are the primary determinants of building block specificity in polyketide biosynthesis and are therefore attractive targets for protein engineering. Thus far, investigations into the fundamental biochemical properties of AT domains have been hampered by the inability to produce these enzymes as self-standing polypeptides. Here we describe an alternative, generally applicable strategy for overexpression and analysis of AT domains from modular PKSs as truncated didomain proteins (approximately 60 kDa). Recently, we reported the expression and reconstitution of the loading didomain of 6-deoxyerythronolide B synthase (Lau, J., Cane, D. E., and Khosla, C. (2000) Biochemistry 39, 10514-20). By replacing the AT domain of this protein with a methylmalonyl-CoA specific AT domain from module 6 of the 6-deoxyerythronolide B synthase, or alternatively a malonyl-CoA specific AT domain from module 2 of the rapamycin synthase, each of these extender unit AT domains could be overproduced and purified to homogeneity. Using acyl-CoA substrates as acyl group donors and N-acetylcysteamine as the thiol acceptor, we devised a steady-state kinetic assay to probe the properties of these three didomain proteins and selected mutants. Propionyl-CoA was the preferred substrate of the loading didomain, although acetyl- and butyryl-CoA were also accepted with approximately 40-fold-lower specificity. In contrast to the relatively relaxed specificity of the loading AT domain, the methylmalonyl- and malonyl-specific AT domains had high specificity (>1000-fold) toward their natural substrates. The acyl transfer reaction was inhibited by coenzyme A (CoASH) with both a competitive and a noncompetitive component. Use of an exogenous holo-acyl carrier protein (ACP) as an acceptor thiol did not increase the rate of acyl transfer relative to the reaction involving N-acetylcysteamine, suggesting that either the on-rate of the acyl group is rate-limiting or that the apo-ACP component of the didomain protein precludes effective docking of a second ACP onto the AT active site. Mutation of Trp-222 in the loading AT domain to an Arg residue that is universally conserved in all extender unit AT domains failed to enable the loading AT domain to accept methylmalonyl-CoA as an alternative substrate. In contrast, mutation of the equivalent Arg residue in an extender AT domain resulted in a protein with no activity. Together, these results provide a foundation for future structural and mechanistic investigations into the properties of AT domains of modular PKSs.