Evidence for DNA phosphate backbone alkylation and cleavage by pyrrolo[1,2-a]benzimidazoles: small molecules capable of causing base-pair-specific phosphodiester bond hydrolysis.

This report presents evidence that a reduced pyrrolo[1,2-a]benzimidazole (PBI) cleaves DNA as a result of phosphate alkylation followed by hydrolysis of the resulting phosphate triester. The base-pair specificity of the phosphate alkylation results from Hoogsteen-type hydrogen bonding of the reduced PBI in the major groove at only A.T and G.C base pairs. Alkylated phosphates were detected by 31P NMR and the cleavage products were detected by 1H NMR and HPLC. Evidence is also presented that a reduced PBI interacts with DNA in the major groove rather than in the minor groove or by intercalation.

Characterization of DNA Strand Transfer Promoted by Mycobacterium smegmatis RecA Reveals Functional Diversity with Mycobacterium tuberculosis RecA†

The RecA-like proteins constitute a group of DNA strand transfer proteins ubiquitous in eubacteria, eukarya, and archaea. However, the functional relationship among RecA proteins is poorly understood. For instance, Mycobacterium tuberculosis RecA is synthesized as a large precursor, which undergoes an unusual protein-splicing reaction to generate an active form. Whereas the precursor was inactive, the active form promoted DNA strand transfer less efficiently compared to EcRecA. Furthermore, gene disruption studies have indicated that the frequencies of allele exchange are relatively lower in Mycobacterium tuberculosis compared to Mycobacterium smegmatis. The mechanistic basis and the factors that contribute to differences in allele exchange remain to be understood. Here, we show that the extent of DNA strand transfer promoted by the M. smegmatis RecA in vitro differs significantly from that of M. tuberculosis RecA. Importantly, M. smegmatis RecA by itself was unable to promote strand transfer, but cognate or noncognate SSBs rendered it efficient even when added prior to RecA. In the presence of SSB, MsRecA or MtRecA catalyzed strand transfer between ssDNA and varying lengths of linear duplex DNA with distinctly different pH profiles. The factors that were able to suppress the formation of DNA networks greatly stimulated strand transfer reactions promoted by MsRecA or MtRecA. Although the rate and pH profiles of dATP hydrolysis catalyzed by MtRecA and MsRecA were similar, only MsRecA was able to couple dATP hydrolysis to DNA strand transfer. Together, these results provide insights into the functional diversity in DNA strand transfer promoted by RecA proteins of pathogenic and nonpathogenic species of mycobacteria.

DNA-induced conformational changes in RecA protein. Evidence for structural heterogeneity among nucleoprotein filaments and implications for homologous pairing

We have used circular dichroism as a probe to characterize the solution conformational changes in RecA protein upon binding to DNA. This approach revealed that RecA protein acquires significant amounts of alpha-helix upon interaction with DNA. These observations, consistent with the data from crystal structure (Story, R. M., Weber, I., and Steitz, T. (1992) Nature 355, 318-325), support the notion that some basic domains including the DNA binding motifs of RecA protein are unstructured and might contribute to the formation of alpha-helix. A comparison of nucleoprotein filaments comprised of RecA protein and a variety of DNA substrates revealed important structural heterogeneity. The most significant difference was observed with poly(dG). poly(dC) and related polymers, rich in GC sequences, which induced minimal amounts of alpha-helix in RecA protein. The magnitude of induction of alpha-helix in RecA protein, which occurred concomitant with the production of ternary complexes, was 2-fold higher with homologous than heterologous duplex DNA. Most importantly, the stimulation of ATP hydrolysis by high salt coincided with that of the induction of alpha-helix in RecA protein. These conformational differences provide a basis for thinking about the biochemical and structural transitions that RecA protein experiences during the formal steps of presynapsis, recognition, and alignment of homologous sequences.

Functional Analysis of DNA Replication Fork Reversal Catalyzed by Mycobacterium tuberculosis RuvAB Proteins

Initially discovered in Escherichia coli, RuvAB proteins are ubiquitous in bacteria and play a dual role as molecular motor proteins responsible for branch migration of the Holliday junction(s) and reversal of stalled replication forks. Despite mounting genetic evidence for a crucial role of RuvA and RuvB proteins in reversal of stalled replication forks, the mechanistic aspects of this process are still not fully understood. Here, we elucidate the ability of Mycobacterium tuberculosis RuvAB (MtRuvAB) complex to catalyze the reversal of replication forks using a range of DNA replication fork substrates. Our studies show that MtRuvAB, unlike E. coli RuvAB, is able to drive replication fork reversal via the formation of Holliday junction intermediates, suggesting that RuvAB-catalyzed fork reversal involves concerted unwinding and annealing of nascent leading and lagging strands. We also demonstrate the reversal of replication forks carrying hemi-replicated DNA, indicating that MtRuvAB complex-catalyzed fork reversal is independent of symmetry at the fork junction. The fork reversal reaction catalyzed by MtRuvAB is coupled to ATP hydrolysis, is processive, and culminates in the formation of an extended reverse DNA arm. Notably, we found that sequence heterology failed to impede the fork reversal activity of MtRuvAB. We discuss the implications of these results in the context of recognition and processing of varied types of replication fork structures by RuvAB proteins.

Mammalian RAD51 paralogs protect nascent DNA at stalled forks and mediate replication restart

Mammalian RAD51 paralogs are implicated in the repair of collapsed replication forks by homologous recombination. However, their physiological roles in replication fork maintenance prior to fork collapse remain obscure. Here, we report on the role of RAD51 paralogs in short-term replicative stress devoid of DSBs. We show that RAD51 paralogs localize to nascent DNA and common fragile sites upon replication fork stalling. Strikingly, RAD51 paralogs deficient cells exhibit elevated levels of 53BP1 nuclear bodies and increased DSB formation, the latter being attributed to extensive degradation of nascent DNA at stalled forks. RAD51C and XRCC3 promote the restart of stalled replication in an ATP hydrolysis dependent manner by disengaging RAD51 and other RAD51 paralogs from the halted forks. Notably, we find that Fanconi anemia (FA)-like disorder and breast and ovarian cancer patient derived mutations of RAD51C fails to protect replication fork, exhibit under-replicated genomic regions and elevated micro-nucleation. Taken together, RAD51 paralogs prevent degradation of stalled forks and promote the restart of halted replication to avoid replication fork collapse, thereby maintaining genomic integrity and suppressing tumorigenesis.

Mutations in the nucleotide binding and hydrolysis domains of Helicobacter pylori MutS2 lead to altered biochemical activities and inactivation of its in vivo function

Background: Helicobacter pylori MutS2 (HpMutS2), an inhibitor of recombination during transformation is a non-specific nuclease with two catalytic sites, both of which are essential for its anti-recombinase activity. Although HpMutS2 belongs to a highly conserved family of ABC transporter ATPases, the role of its ATP binding and hydrolysis activities remains elusive. Results: To explore the putative role of ATP binding and hydrolysis activities of HpMutS2 we specifically generated point mutations in the nucleotide-binding Walker-A (HpMutS2-G338R) and hydrolysis Walker-B (HpMutS2-E413A) domains of the protein. Compared to wild-type protein, HpMutS2-G338R exhibited similar to 2.5-fold lower affinity for both ATP and ADP while ATP hydrolysis was reduced by similar to 3-fold. Nucleotide binding efficiencies of HpMutS2-E413A were not significantly altered; however the ATP hydrolysis was reduced by similar to 10-fold. Although mutations in the Walker-A and Walker-B motifs of HpMutS2 only partially reduced its ability to bind and hydrolyze ATP, we demonstrate that these mutants not only exhibited alterations in the conformation, DNA binding and nuclease activities of the protein but failed to complement the hyper-recombinant phenotype displayed by mutS2-disrupted strain of H. pylori. In addition, we show that the nucleotide cofactor modulates the conformation, DNA binding and nuclease activities of HpMutS2. Conclusions: These data describe a strong crosstalk between the ATPase, DNA binding, and nuclease activities of HpMutS2. Furthermore these data show that both, ATP binding and hydrolysis activities of HpMutS2 are essential for the in vivo anti-recombinase function of the protein.

Expanding the repertoire of DNA-mediated signaling in DNA repair

DNA damage is extremely detrimental to the cell and must be repaired to protect the genome. DNA is capable of conducting charge through the overlapping π-orbitals of stacked bases; this phenomenon is extremely sensitive to the integrity of the π-stack, as perturbations attenuate DNA charge transport (CT). Based on the E. coli base excision repair (BER) proteins EndoIII and MutY, it has recently been proposed that redox-active proteins containing metal clusters can utilize DNA CT to signal one another to locate sites of DNA damage.

To expand our repertoire of proteins that utilize DNA-mediated signaling, we measured the DNA-bound redox potential of the nucleotide excision repair (NER) helicase XPD from Sulfolobus acidocaldarius. A midpoint potential of 82 mV versus NHE was observed, resembling that of the previously reported BER proteins. The redox signal increases in intensity with ATP hydrolysis in only the WT protein and mutants that maintain ATPase activity and not for ATPase-deficient mutants. The signal increase correlates directly with ATP activity, suggesting that DNA-mediated signaling may play a general role in protein signaling. Several mutations in human XPD that lead to XP-related diseases have been identified; using SaXPD, we explored how these mutations, which are conserved in the thermophile, affect protein electrochemistry.

To further understand the electrochemical signaling of XPD, we studied the yeast S. cerevisiae Rad3 protein. ScRad3 mutants were incubated on a DNA-modified electrode and exhibited a similar redox potential to SaXPD. We developed a haploid strain of S. cerevisiae that allowed for easy manipulation of Rad3. In a survival assay, the ATPase- and helicase-deficient mutants show little survival, while the two disease-related mutants exhibit survival similar to WT. When both a WT and G47R (ATPase/helicase deficient) strain were challenged with different DNA damaging agents, both exhibited comparable survival in the presence of hydroxyurea, while with methyl methanesulfonate and camptothecin, the G47R strain exhibits a significant change in growth, suggesting that Rad3 is involved in repairing damage beyond traditional NER substrates. Together, these data expand our understanding of redox-active proteins at the interface of DNA repair.

Binding site size limitations of imidazole-pyrrole polyamides for recognition in the minor groove of DNA

The discovery that the three ring polyamide Im-Py-Py-Dp containing imidazole (Im) and pyrrole (Py) carboxamides binds the DNA sequence 5'-(A,T)G(A,T)C(A,T)-3' as an antiparallel dimer offers a new model for the design of ligands for specific recognition of sequences in the minor groove containing both G,C and A,T base pairs. In Chapter 2, experiments are described in which the sequential addition of five N- methylpyrrolecarboxamides to the imidazole-pyrrole polyamide Im-Py-Py-Dp affords a series of six homologous polyamides, Im-(Py)_2-7-Dp, that differ in the size of their binding site, apparent first order binding affinity, and sequence specificity. These results demonstrate that DNA sequences up to nine base pairs in length can be specifically recognized by imidazole-pyrrole polyamides containing three to seven rings by 2:1 polyamide-DNA complex formation in the minor groove. Recognition of a nine base pair site defines the new lower limit of the binding site size that can be recognized by polyamides containing exclusively imidazole and pyrrolecarboxamides. The results of this study should provide useful guidelines for the design of new polyamides that bind longer DNA sites with enhanced affinity and specificity.

In Chapter 3 the design and synthesis of the hairpin polyamide Im-Py-Im-Py-γ-Im- Py-Im-Py-Dp is described. Quantitative DNase I footprint titration experiments reveal that Im-Py-Im-Py-γ-Im-Py-Im-Py-Dp binds six base pair 5'-(A,T)GCGC(A,T)-3' sequences with 30-fold higher affinity than the unlinked polyamide Im-Py-Im-Py-Dp. The hairpin polyamide does not discriminate between A•T and T•A at the first and sixth positions of the binding site as three sites 5'-TGCGCT-3', 5'-TGCGCA-3', and 5 'AGCGCT- 3' are bound with similar affinity. However, Im-Py-Im-Py-γ-Im-Py-Im-PyDp is specific for and discriminates between G•C and C•G base pairs in the 5'-GCGC-3' core as evidenced by lower affinities for the mismatched sites 5'-AACGCA-3', 5'- TGCGTT-3', 5'-TGCGGT-3', and 5'-ACCGCT-3'.

In Chapter 4, experiments are described in which a kinetically stable hexa-aza Schiff base La³⁺ complex is covalently attached to a Tat(49-72) peptide which has been shown to bind the HIV-1 TAR RNA sequence. Although these metallo-peptides cleave TAR site-specifically in the hexanucleotide loop to afford products consistent with hydrolysis, a series of control experiments suggests that the observed cleavage is not caused by a sequence-specifically bound Tat(49-72)-La(L)³⁺ peptide.

DNA-mediated charge transport signaling within the cell

DNA possesses the curious ability to conduct charge longitudinally through the π-stacked base pairs that reside within the interior of the double helix. The rate of charge transport (CT) through DNA has a shallow distance dependence. DNA CT can occur over at least 34 nm, a very long molecular distance. Lastly, DNA CT is exquisitely sensitive to disruptions, such as DNA damage, that affect the dynamics of base-pair stacking. Many DNA repair and DNA-processing enzymes are being found to contain 4Fe-4S clusters. These co-factors have been found in glycosylases, helicases, helicase-nucleases, and even enzymes such as DNA polymerase, RNA polymerase, and primase across the phylogeny. The role of these clusters in these enzymes has remained elusive. Generally, iron-sulfur clusters serve redox roles in nature since, formally, the cluster can exist in multiple oxidation states that can be accessed within a biological context. Taken together, these facts were used as a foundation for the hypothesis that DNA-binding proteins with 4Fe-4S clusters utilize DNA-mediated CT as a means to signal one another to scan the genome as a first step in locating the subtle damage that occurs within a sea of undamaged bases within cells.

Herein we describe a role for 4Fe-4S clusters in DNA-mediated charge transport signaling among EndoIII, MutY, and DinG, which are from distinct repair pathways in E. coli. The DinG helicase is an ATP-dependent helicase that contains a 4Fe-4S cluster. To study the DNA-bound redox properties of DinG, DNA-modified electrochemistry was used to show that the 4Fe-4S cluster of DNA-bound DinG is redox-active at cellular potentials, and shares the 80 mV vs. NHE redox potential of EndoIII and MutY. ATP hydrolysis by DinG increases the DNA-mediated redox signal observed electrochemically, likely reflecting better coupling of the 4Fe-4S cluster to DNA while DinG unwinds DNA, which could have interesting biological implications. Atomic force microscopy experiments demonstrate that DinG and EndoIII cooperate at long range using DNA charge transport to redistribute to regions of DNA damage. Genetics experiments, moreover, reveal that this DNA-mediated signaling among proteins also occurs within the cell and, remarkably, is required for cellular viability under conditions of stress. Knocking out DinG in CC104 cells leads to a decrease in MutY activity that is rescued by EndoIII D138A, but not EndoIII Y82A. DinG, thus, appears to help MutY find its substrate using DNA-mediated CT, but do MutY or EndoIII aid DinG in a similar way? The InvA strain of bacteria was used to observe DinG activity, since DinG activity is required within InvA to maintain normal growth. Silencing the gene encoding EndoIII in InvA results in a significant growth defect that is rescued by the overexpression of RNAseH, a protein that dismantles the substrate of DinG, R-loops. This establishes signaling between DinG and EndoIII. Furthermore, rescue of this growth defect by the expression of EndoIII D138A, the catalytically inactive but CT-proficient mutant of EndoIII, is also observed, but expression of EndoIII Y82A, which is CT-deficient but enzymatically active, does not rescue growth. These results provide strong evidence that DinG and EndoIII utilize DNA-mediated signaling to process DNA damage. This work thus expands the scope of DNA-mediated signaling within the cell, as it indicates that DNA-mediated signaling facilitates the activities of DNA repair enzymes across the genome, even for proteins from distinct repair pathways.

In separate work presented here, it is shown that the UvrC protein from E. coli contains a hitherto undiscovered 4Fe-4S cluster. A broad shoulder at 410 nm, characteristic of 4Fe-4S clusters, is observed in the UV-visible absorbance spectrum of UvrC. Electron paramagnetic resonance spectroscopy of UvrC incubated with sodium dithionite, reveals a spectrum with the signature features of a reduced, [4Fe-4S]+1, cluster. DNA-modified electrodes were used to show that UvrC has the same DNA-bound redox potential, of ~80 mV vs. NHE, as EndoIII, DinG, and MutY. Again, this means that these proteins are capable of performing inter-protein electron transfer reactions. Does UvrC use DNA-mediated signaling to facilitate the repair of its substrates?

UvrC is part of the nucleotide excision repair (NER) pathway in E. coli and is the protein within the pathway that performs the chemistry required to repair bulky DNA lesions, such as cyclopyrimidine dimers, that form as a product of UV irradiation. We tested if UvrC utilizes DNA-mediated signaling to facilitate the efficient repair of UV-induced DNA damage products by helping UvrC locate DNA damage. The UV sensitivity of E. coli cells lacking DinG, a putative signaling partner of UvrC, was examined. Knocking out DinG in E. coli leads to a sensitivity of the cells to UV irradiation. A 5-10 fold reduction in the amount of cells that survive after irradiation with 90 J/m2 of UV light is observed. This is consistent with the hypothesis that UvrC and DinG are signaling partners, but is this signaling due to DNA-mediated CT? Complementing the knockout cells with EndoIII D138A, which can also serve as a DNA CT signaling partner, rescues cells lacking DinG from UV irradiation, while complementing the cells with EndoIII Y82A shows no rescue of viability. These results indicate that there is cross-talk between the NER pathway and DinG via DNA-mediated signaling. Perhaps more importantly, this work also establishes that DinG, EndoIII, MutY, and UvrC comprise a signaling network that seems to be unified by the ability of these proteins to perform long range DNA-mediated CT signaling via their 4Fe-4S clusters.

Studies on the stability of polyazamacrocycle-Eu (Tb) by potentiometry and its catalytic hydrolysis on phosphate diester

The stability constants and species distributions of complexes of two lanthanide ions, Eu (III) and Tb(III), with a macrocyclic ligand, 3,6, 9, 17 20, 23-hexaazo-29, 30-dihydroxy-13, 27-dimethyl-tricylco-[23,3,1,1(11,15)] triaconta-1 (28) 11,13,15 (30), 25 26-hexane (BDBPH), in 1: 1 and 2: 1 system, were determined potentiometrically in 50% ethanol solution, at 35.0 degrees C and I = 0.100 mol/L (KCl). The two metal ions could form deprotonated mono- or dinuclear complexes with BDBPH with high stability after the three protons of the ligand completely neutralized. At higher pH values, Eu(M) could not form hydroxo complexes with BDBPH, while Tb(III) could form hydroxo complexes in the types of M2L(OH) M2L(OH)(2) and M2L (OH)(2). The kinetic study on the hydrolysis reaction of his (4-nitrophenyl) phosphate (BNPP) catalyzed by Tb-BDBPH system (2:1) was carried out in aqueous solution (pH 7.0 similar to 10.0) at 35 degrees C with I = 0.1000 mol/L (KCl). The second-order rate constant k(BNPP) (2.3 x 10(-3) (mol/L)(-1)center dot s(-1)) was determined. The dinuclear monohydroxo species, L-Tb-2-OH, is kinetically active species.

Use of lanthanide metal complexes for the hydrolysis of 3 ',5 '- and 2 ',3 ' cyclic nucleotides

Lutetium(III) and lanthanum(III) complexes of 2-carboxyethylgermanium sesquioxide (Ge-132) can hydrolyze the phosphodiester linkage of 3',5'-cyclic adenosine monophosphate (cAMP), 3',5'-cyclic deoxyadenosine monophosphate (dcAMP) and 2',3'-cyclic adenosine monophosphate (2',3'-cAMP). Both cAMP and dcAMP are hydrolyzed with high selectivity, yielding predominantly 3'-monophosphates. 2',3'-cAMP is converted to 3'-AMP and 2'-AMP, the ratio of 3'-AMP to 2'-AMP produced being 1.4.

Lanthanide metal complexes for the hydrolysis of ribonucleoside 3',5'-cyclic phosphate and deoxyribonucleoside 3',5'-cyclic phosphate

Ytterbium(III) and praseodymium(III) complexes of 2-carboxyethylgermanium sesquioxide (Ge-132) can hydrolyze the phosphodiester linkage of 3',5'-cyclic adenosine monophosphate (cAMP) and 3',5'-cyclic deoxyadenosine monophosphate (dcAMP). Both cAMP and dcAMP are hydrolyzed with high selectivity, yielding predominantly 3'-monophosphates. The selectivity and activity for hydrolyzing cAMP and dcAMP by lanthanide metal(III) complexes and lanthanide metal ions are compared.

Hydrolysis of adenosine monophosphate and guanosine monophosphate by lanthanides

The hydrolysis of adenosine-5'-monophosphate(5'-AMP) and guanosine-5'-monophosphate(5'-GMP) by lanthanides was investigated. 5'-AMP and 5'-GMP was efficiently hydrolyzed by cerium(III) chloride under air at pH 9 and 37 degrees C, and other lanthanides (III) showed less efficiency at the same condition. The hydrolysis rate of 5'-AMP by cerium was greater than that of 5'-GMP. UV spectra showed that Ce(III) was oxidized to Ce(IV) in the reaction mixture. The active species for the hydrolysis of 5'-AMP and 5'-GMP was ascribed to the Ce(IV) hydroxide cluster in the reaction mixture.

Regions of the alpha 1-adrenergic receptor involved in coupling to phosphatidylinositol hydrolysis and enhanced sensitivity of biological function.

Regions of the hamster alpha 1-adrenergic receptor (alpha 1 AR) that are important in GTP-binding protein (G protein)-mediated activation of phospholipase C were determined by studying the biological functions of mutant receptors constructed by recombinant DNA techniques. A chimeric receptor consisting of the beta 2-adrenergic receptor (beta 2AR) into which the putative third cytoplasmic loop of the alpha 1AR had been placed activated phosphatidylinositol metabolism as effectively as the native alpha 1AR, as did a truncated alpha 1AR lacking the last 47 residues in its cytoplasmic tail. Substitutions of beta 2AR amino acid sequence in the intermediate portions of the third cytoplasmic loop of the alpha 1AR or at the N-terminal portion of the cytoplasmic tail caused marked decreases in receptor coupling to phospholipase C. Conservative substitutions of two residues in the C terminus of the third cytoplasmic loop (Ala293----Leu, Lys290----His) increased the potency of agonists for stimulating phosphatidylinositol metabolism by up to 2 orders of magnitude. These data indicate (i) that the regions of the alpha 1AR that determine coupling to phosphatidylinositol metabolism are similar to those previously shown to be involved in coupling of beta 2AR to adenylate cyclase stimulation and (ii) that point mutations of a G-protein-coupled receptor can cause remarkable increases in sensitivity of biological response.

Regulation of human dUTPase gene expression and p53-mediated transcriptional repression in response to oxaliplatin-induced DNA damage

Deoxyuridine triphosphate nucleotidohydrolase (dUTPase) catalyzes the hydrolysis of dUTP to dUMP and PPi. Although dUTP is a normal intermediate in DNA synthesis, its accumulation and misincorporation into DNA is lethal. Importantly, uracil misincorporation is a mechanism of cytotoxicity induced by fluoropyrimidine chemotherapeutic agents including 5-fluorouracil (5-FU) and elevated expression of dUTPase is negatively correlated with clinical response to 5-FU-therapy. In this study we performed the first functional characterization of the dUTPase promoter and demonstrate a role for E2F-1 and Sp1 in driving dUTPase expression. We establish a direct role for both mutant and wild-type forms of p53 in modulating dUTPase promoter activity. Treatment of HCT116 p53(+/+) cells with the DNA-damaging agent oxaliplatin induced a p53-dependent transcriptional downregulation of dUTPase not observed in the isogenic null cell line. Oxaliplatin treatment induced enrichment of p53 at the dUTPase promoter with a concomitant reduction in Sp1. The suppression of dUTPase by oxaliplatin promoted increased levels of dUTP that was enhanced by subsequent addition of fluoropyrimidines. The novel observation that oxaliplatin downregulates dUTPase expression may provide a mechanistic basis contributing to the synergy observed between 5-FU and oxaliplatin in the clinic. Furthermore, these studies provide the first evidence of a direct transcriptional link between the essential enzyme dUTPase and the tumor suppressor p53.