Gregory Tombline

BCR/ABL Regulates Mammalian RecA Homologs, Resulting in Drug Resistance

RAD51 is one of six mitotic human homologs of the E. coli RecA protein (RAD51-Paralogs) that play a central role in homologous recombination and repair of DNA double-strand breaks (DSBs). Here we demonstrate that RAD51 is important for resistance to cisplatin and mitomycin C in cells expressing the BCR/ABL oncogenic tyrosine kinase. BCR/ABL significantly enhances the expression of RAD51 and several RAD51-Paralogs. RAD51 overexpression is mediated by a STAT5-dependent transcription as well as by inhibition of caspase-3-dependent cleavage. Phosphorylation of the RAD51 Tyr-315 residue by BCR/ABL appears essential for enhanced DSB repair and drug resistance. Induction of the mammalian RecA homologs establishes a unique mechanism for DNA damage resistance in mammalian cells transformed by an oncogenic tyrosine kinase.

Biochemical Characterization of the Human RAD51 Protein I. ATP HYDROLYSIS

The prototypical bacterial RecA protein promotes recombination/repair by catalyzing strand exchange between homologous DNAs. While the mechanism of strand exchange remains enigmatic, ATP-induced cooperativity between RecA protomers is critical for its function. A human RecA homolog, human RAD51 protein (hRAD51), facilitates eukaryotic recombination/repair, although its ability to hydrolyze ATP and/or promote strand exchange appears distinct from the bacterial RecA. We have quantitatively examined the hRAD51 ATPase. The catalytic efficiency (k cat/K m ) of the hRAD51 ATPase was ∼50-fold lower than the RecA ATPase. Altering the ratio of DNA/hRAD51 and including salts that stimulate DNA strand exchange (ammonium sulfate and spermidine) were found to affect the catalytic efficiency of hRAD51. The average site size of hRAD51 was determined to be ∼3 nt (bp) for both single-stranded and double-stranded DNA. Importantly, hRAD51 lacks the magnitude of ATP-induced cooperativity that is a hallmark of RecA. Together, these results suggest that hRAD51 may be unable to coordinate ATP hydrolysis between neighboring protomers.

Synergy between conserved ABC signature Ser residues in P-glycoprotein catalysis.

Functional roles of the two ABC signature sequences (“LSGGQ”) in the N- and C-terminal nucleotide binding domains of P-glycoprotein were studied by mutating the conserved Ser residues to Ala. The two single mutants (S528A; S1173A) each impaired ATPase activity mildly, and showed generally symmetrical effects on function, consistent with equivalent mechanistic roles of the two nucleotide sites. Synergy between the two mutations when combined was remarkable and resulted in strong catalytic impairment. The Ser residues are not involved significantly in MgATP- or MgADP-binding or in interdomain communication between catalytic sites and drug binding sites. Retention of product MgADP is not the cause of reduced turnover. Mutation of Ser to Ala reduced the strength of interaction with the chemical transition state specifically, as shown by vanadate-ADP and beryllium fluoride-ADP trapping experiments. Therefore, the two conserved ABC signature motif Ser residues of P-glycoprotein cooperatively accelerate ATP hydrolysis via chemical transition state interaction. Because the transition state complex is currently believed to form in the dimerized state of the nucleotide binding domains, one may also conclude that both Ser-OH are necessary for correct formation of the dimer state.

Rhodamine Inhibitors of P-glycoprotein: An Amide/Thioamide “Switch” for ATPase Activity

We have examined 46 tetramethylrosamine/rhodamine derivatives with structural diversity in the heteroatom of the xanthylium core, the amino substituents of the 3- and 6-positions, and the alkyl, aryl, or heteroaryl group at the 9-substituent. These compounds were examined for affinity and ATPase stimulation in isolated MDR3 CL P-gp and human P-gp-His(10), for their ability to promote uptake of calcein AM and vinblastine in multidrug-resistant MDCKII-MDR1 cells, and for transport in monolayers of MDCKII-MDR1 cells. Thioamide 31-S gave K(M) of 0.087 microM in human P-gp. Small changes in structure among this set of compounds affected affinity as well as transport rate (or flux) even though all derivatives examined were substrates for P-gp. With isolated protein, tertiary amide groups dictate high affinity and high stimulation while tertiary thioamide groups give high affinity and inhibition of ATPase activity. In MDCKII-MDR1 cells, the tertiary thioamide-containing derivatives promote uptake of calcein AM and have very slow passive, absorptive, and secretory rates of transport relative to transport rates for tertiary amide-containing derivatives. Thioamide 31-S promoted uptake of calcein AM and inhibited efflux of vinblastine with IC(50)s of approximately 2 microM in MDCKII-MDR1 cells.

Biochemical Characterization of the Human RAD51 Protein III. MODULATION OF DNA BINDING BY ADENOSINE NUCLEOTIDES

Adenosine nucleotides affect the ability of RecA·single-stranded DNA (ssDNA) nucleoprotein filaments to cooperatively assume and maintain an extended structure that facilitates DNA pairing during recombination. Here we have determined that ADP and ATP/ATPγS affect the DNA binding and aggregation properties of the human RecA homolog human RAD51 protein (hRAD51). These studies have revealed significant differences between hRAD51 and RecA. In the presence of ATPγS, RecA forms a stable complex with ssDNA, while the hRAD51 ssDNA complex is destabilized. Conversely, in the presence of ADP and ATP, the RecA ssDNA complex is unstable, while the hRAD51 ssDNA complex is stabilized. We identified two hRAD51·ssDNA binding forms by gel shift analysis, which were distinct from a well defined RecA·ssDNA binding form. The available evidence suggests that a low molecular weight hRAD51·ssDNA binding form (hRAD51·ssDNAlow) correlates with active ADP and ATP processing. A high molecular weight hRAD51·ssDNA aggregate (hRAD51·ssDNAhigh) appears to correlate with a form that fails to process ADP and ATP. Our data are consistent with the notion that hRAD51 is unable to appropriately coordinate ssDNA binding with adenosine nucleotide processing. These observations suggest that other factors may assist hRAD51 in order to mirror RecA recombinational function.

Biochemical Characterization of the Human RAD51 Protein II. ADENOSINE NUCLEOTIDE BINDING AND COMPETITION

RecA mediated homologous recombination requires cooperative ATP binding and hydrolysis to assume and maintain an active, extended DNA-protein (nucleoprotein) filament. Human RAD51 protein (hRAD51) lacks the magnitude of ATP-induced cooperativity and catalytic efficiency displayed by RecA. Here, we examined hRAD51 binding and ATPase inhibition pattern by ADP and ATP/adenosine 5′-O-(thiotriphosphate) (ATPγS). hRAD51 fully saturates with ATP/ATPγS regardless of DNA cofactor (K D ≈ 5 μm; 1 ATP/1 hRAD51). The binding of ADP to hRAD51 appeared bimodal. The first mode was identical to ATP/ATPγS binding (K app1 ≈ 3 μm; 1 ADP/1 hRAD51), while a second mode occurred at elevated ADP concentrations (K app2 ≥ 125 μm; >1 ADP/1 hRAD51). We could detect ADP → ATP exchange in the high affinity ADP binding mode (K app1) but not the low affinity binding mode (K app2). At low ATP concentrations (<0.3 mm), ADP and ATPγS competitively inhibit the hRAD51 ATPase (K m (app) >K m ). However, at high ATP (>0.3 mm), the hRAD51 ATPase was stimulated by concentrations of ATPγS that were 20-fold above the K D . Ammonium sulfate plus spermidine decreased the affinity of hRAD51 for ADP substantially (∼10-fold) and ATP modestly (∼3-fold). Our results suggest that ATP binding is not rate-limiting but that the inability to sustain an active nucleoprotein filament probably restricts the hRAD51 ATPase.

JNK Phosphorylates SIRT6 to Stimulate DNA Double-Strand Break Repair in Response to Oxidative Stress by Recruiting PARP1 to DNA Breaks

SUMMARY The accumulation of damage caused by oxidative stress has been linked to aging and to the etiology of numerous age-related diseases. The longevity gene, sirtuin 6 (SIRT6), promotes genome stability by facilitating DNA repair, especially under oxidative stress conditions. Here we uncover the mechanism by which SIRT6 is activated by oxidative stress to promote DNA double-strand break (DSB) repair. We show that the stress-activated protein kinase, c-Jun N-terminal kinase (JNK), phosphorylates SIRT6 on serine 10 in response to oxidative stress. This post-translational modification facilitates the mobilization of SIRT6 to DNA damage sites and is required for efficient recruitment of poly (ADP-ribose) polymerase 1 (PARP1) to DNA break sites and for efficient repair of DSBs. Our results demonstrate a post-translational mechanism regulating SIRT6, and they provide the link between oxidative stress signaling and DNA repair pathways that may be critical for hormetic response and longevity assurance.

Pseudomonas aeruginosa PA1006, Which Plays a Role in Molybdenum Homeostasis, Is Required for Nitrate Utilization, Biofilm Formation, and Virulence

Pseudomonas aeruginosa (Pae) is a clinically important opportunistic pathogen. Herein, we demonstrate that the PA1006 protein is critical for all nitrate reductase activities, growth as a biofilm in a continuous flow system, as well as virulence in mouse burn and rat lung model systems. Microarray analysis revealed that ΔPA1006 cells displayed extensive alterations in gene expression including nitrate-responsive, quorum sensing (including PQS production), and iron-regulated genes, as well as molybdenum cofactor and Fe-S cluster biosynthesis factors, members of the TCA cycle, and Type VI Secretion System components. Phenotype Microarray™ profiles of ΔPA1006 aerobic cultures using Biolog plates also revealed a reduced ability to utilize a number of TCA cycle intermediates as well as a failure to utilize xanthine as a sole source of nitrogen. As a whole, these data indicate that the loss of PA1006 confers extensive changes in Pae metabolism. Based upon homology of PA1006 to the E. coli YhhP protein and data from the accompanying study, loss of PA1006 persulfuration and/or molybdenum homeostasis are likely the cause of extensive metabolic alterations that impact biofilm development and virulence in the ΔPA1006 mutant.

Mass Spectrometry Detection of Histidine Phosphorylation on NM23-H1

Phosphorylation is a ubiquitous protein post-translational modification that is intimately involved in most aspects of cellular regulation. Currently, most proteomic analyses are performed with phosphorylation searches for serine, threonine, and tyrosine modifications, as the phosphorylated residues of histidine and aspartic acid are acid labile and thus undetectable with most proteomic methodologies. Here, we present a novel buffer system to show histidine phosphorylation of NM23-H1, the product of the first identified putative human metastasis suppressor gene (NME1), which catalyzes the transfer of the γ-phosphate from nucleoside triphosphates to nucleoside diphosphates. On the basis of a pH titration of LC elution buffers and MS/MS identification, recombinant NM23-H1 subjected to autophosphorylation was shown to contain phosphorylated histidine at residue 118 at pH 5 and 6, with each level giving over 75% peptide coverage for identification. The solvent system presented permits the detection of all five possible phosphorylation moieties. Application of histidine and aspartic acid phosphorylation modifications to proteomic analyses will significantly advance the understanding of phosphorylation relay signaling in cellular regulation, including elucidation of the role of NM23-H1 in metastasis.

ATP occlusion by P-glycoprotein as a surrogate measure for drug coupling.

The multidrug efflux pump P-glycoprotein (Pgp) couples drug transport to ATP hydrolysis. Previously, using a synthetic library of tetramethylrosamine ( TMR) analogues, we observed significant variation in ATPase stimulation ( V m (D)). Concentrations required for half-maximal ATPase stimulation ( K m (D)) correlated with ATP hydrolysis transition-state stabilization and ATP occlusion (EC 50 (D)) at a single site. Herein, we characterize several TMR analogues that elicit modest turnover ( k cat <or= 1-2 s (-1)) compared to verapamil (VER) ( k cat approximately 10 s (-1)). Apparent ATPase activities manifest as nearly equivalent to basal values. In some cases, K m (D) parameters for drug stimulation of ATPase could not be accurately determined, yet these same TMR analogues promoted ATP occlusion at relatively low concentrations ( approximately 0.4-40 microM). Moreover, the TMR analogues competitively inhibited VER-dependent ATPase activity at concentrations similar to those required for ATP occlusion. Finally, the TMR analogues facilitated uptake of calcein-AM into CR1R12 and MDCK-MDR1 cells and are actively transported by Pgp in monolayers of MDCK-MDR1 cells at similarly low concentrations ( approximately 1-20 microM). ADP.V i release kinetics were identical in the presence of the TMR derivatives, VER, or in the absence of drug, suggesting that slow turnover is not likely due to slow release of the ATP hydrolysis products ADP and P i. These data support the partition model in which drug site occupancy converts residual basal ATPase activity to a drug-dependent mechanism even in cases where stimulation appears to be exactly compensatory to basal values. It is noteworthy that when compared to previously reported TMR analogues, subtle modification of the TMR scaffold can confer large differences in ATP turnover.