Human DNA ligases I and III have stand-alone end-joining capability, but differ in ligation efficiency and specificity.

Double-strand DNA breaks (DSBs) are toxic to cells, and improper repair can cause chromosomal abnormalities that initiate and drive cancer progression. DNA ligases III and IV (LIG3, LIG4) have long been credited for repair of DSBs in mammals, but recent evidence suggests that DNA ligase I (LIG1) has intrinsic end-joining (EJ) activity that can compensate for their loss. To test this model, we employed in vitro biochemical assays to compare EJ by LIG1 and LIG3. The ligases join blunt-end and 3'-overhang-containing DNA substrates with similar catalytic efficiency, but LIG1 joins 5'-overhang-containing DNA substrates ∼20-fold less efficiently than LIG3 under optimal conditions. LIG1-catalyzed EJ is compromised at a physiological concentration of Mg2+, but its activity is restored by increased molecular crowding. In contrast to LIG1, LIG3 efficiently catalyzes EJ reactions at a physiological concentration of Mg2+ with or without molecular crowding. Under all tested conditions, LIG3 has greater affinity than LIG1 for DNA ends. Remarkably, LIG3 can ligate both strands of a DSB during a single binding encounter. The weaker DNA binding affinity of LIG1 causes significant abortive ligation that is sensitive to molecular crowding and DNA terminal structure. These results provide new insights into mechanisms of alternative nonhomologous EJ.

Comparison of quartz and sapphire optical chambers for infrared laser sealing of vascular tissues using a reciprocating, side-firing optical fiber: Simulations and experiments.

INTRODUCTION: Infrared (IR) lasers are being tested as an alternative to radiofrequency (RF) and ultrasonic (US) surgical devices for hemostatic sealing of vascular tissues. In previous studies, a side-firing optical fiber with elliptical IR beam output was reciprocated, producing a linear IR laser beam pattern for uniform sealing of blood vessels. Technical challenges include limited field-of-view of vessel position within the metallic device jaws, and matching fiber scan length to variable vessel sizes. A transparent jaw may improve visibility and enable custom treatment. METHODS: Quartz and sapphire square optical chambers (2.7 × 2.7 × 25 [mm3 ] outer dimensions) were tested, capable of fitting into a 5-mm-OD laparoscopic device. A 1470 nm laser was used for optical transmission studies. Razor blade scans and an IR beam profiler acquired fiber (550-µm-core/0.22NA) output beam profiles. Thermocouples recorded peak temperatures and cooling times on internal and external chamber surfaces. Optical fibers with angle polished distal tips delivered 94% of light at a 90° angle. Porcine renal arteries with diameters of 3.4 ± 0.7 mm (n = 13) for quartz and 3.2 ± 0.7 mm (n = 14) for sapphire chambers (p > 0.05), were sealed using 30 W for 5 s. RESULTS: Reflection losses at material/air interfaces were 3.3% and 7.4% for quartz and sapphire. Peak temperatures on the external chamber surface averaged 74 ± 8°C and 73 ± 10°C (p > 0.05). Times to cool down to 37°C measured 13 ± 4 s and 27 ± 7 s (p < 0.05). Vessel burst pressures (BP) averaged 883 ± 393 mmHg and 412 ± 330 mmHg (p < 0.05). For quartz, 13/13 (100%) vessels were sealed (BP > 360 mmHg), versus 9/14 (64%) for sapphire. Computer simulations for the quartz chamber yielded peak temperatures (78°C) and cooling times (16 s) similar to experiments. CONCLUSIONS: Quartz is an inexpensive material for use in a laparoscopic device jaw, providing more consistent vessel seals and faster cooling times than sapphire and current RF and US devices.

LIG1 syndrome mutations remodel a cooperative network of ligand binding interactions to compromise ligation efficiency.

Human DNA ligase I (LIG1) is the main replicative ligase and it also seals DNA breaks to complete DNA repair and recombination pathways. Immune compromised patients harbor hypomorphic LIG1 alleles encoding substitutions of conserved arginine residues, R771W and R641L, that compromise LIG1 activity through poorly defined mechanisms. To understand the molecular basis of LIG1 syndrome mutations, we determined high resolution X-ray structures and performed systematic biochemical characterization of LIG1 mutants using steady-state and pre-steady state kinetic approaches. Our results unveil a cooperative network of plastic DNA-LIG1 interactions that connect DNA substrate engagement with productive binding of Mg2+ cofactors for catalysis. LIG1 syndrome mutations destabilize this network, compromising Mg2+ binding affinity, decreasing ligation efficiency, and leading to elevated abortive ligation that may underlie the disease pathology. These findings provide novel insights into the fundamental mechanism by which DNA ligases engage with a nicked DNA substrate, and they suggest that disease pathology of LIG1 syndrome could be modulated by Mg2+ levels.

Recognition of 1,N2-ethenoguanine by alkyladenine DNA glycosylase is restricted by a conserved active-site residue.

The adenine, cytosine, and guanine bases of DNA are susceptible to alkylation by the aldehyde products of lipid peroxidation and by the metabolic byproducts of vinyl chloride pollutants. The resulting adducts spontaneously cyclize to form harmful etheno lesions. Cells employ a variety of DNA repair pathways to protect themselves from these pro-mutagenic modifications. Human alkyladenine DNA glycosylase (AAG) is thought to initiate base excision repair of both 1,N6-ethenoadenine (ϵA) and 1,N2-ethenoguanine (ϵG). However, it is not clear how AAG might accommodate ϵG in an active site that is complementary to ϵA. This prompted a thorough investigation of AAG-catalyzed excision of ϵG from several relevant contexts. Using single-turnover and multiple-turnover kinetic analyses, we found that ϵG in its natural ϵG·C context is very poorly recognized relative to ϵA·T. Bulged and mispaired ϵG contexts, which can form during DNA replication, were similarly poor substrates for AAG. Furthermore, AAG could not recognize an ϵG site in competition with excess undamaged DNA sites. Guided by previous structural studies, we hypothesized that Asn-169, a conserved residue in the AAG active-site pocket, contributes to discrimination against ϵG. Consistent with this model, the N169S variant of AAG was 7-fold more active for excision of ϵG compared with the wildtype (WT) enzyme. Taken together, these findings suggest that ϵG is not a primary substrate of AAG, and that current models for etheno lesion repair in humans should be revised. We propose that other repair and tolerance mechanisms operate in the case of ϵG lesions.

Transient kinetic analysis of oxidative dealkylation by the direct reversal DNA repair enzyme AlkB.

AlkB is a bacterial Fe(II)- and 2-oxoglutarate-dependent dioxygenase that repairs a wide range of alkylated nucleobases in DNA and RNA as part of the adaptive response to exogenous nucleic acid-alkylating agents. Although there has been longstanding interest in the structure and specificity of Escherichia coli AlkB and its homologs, difficulties in assaying their repair activities have limited our understanding of their substrate specificities and kinetic mechanisms. Here, we used quantitative kinetic approaches to determine the transient kinetics of recognition and repair of alkylated DNA by AlkB. These experiments revealed that AlkB is a much faster alkylation repair enzyme than previously reported and that it is significantly faster than DNA repair glycosylases that recognize and excise some of the same base lesions. We observed that whereas 1,N6-ethenoadenine can be repaired by AlkB with similar efficiencies in both single- and double-stranded DNA, 1-methyladenine is preferentially repaired in single-stranded DNA. Our results lay the groundwork for future studies of AlkB and its human homologs ALKBH2 and ALKBH3.

Expansion of base excision repair compensates for a lack of DNA repair by oxidative dealkylation in budding yeast.

The Mag1 and Tpa1 proteins from budding yeast (Saccharomyces cerevisiae) have both been reported to repair alkylation damage in DNA. Mag1 initiates the base excision repair pathway by removing alkylated bases from DNA, and Tpa1 has been proposed to directly repair alkylated bases as does the prototypical oxidative dealkylase AlkB from Escherichia coli However, we found that in vivo repair of methyl methanesulfonate (MMS)-induced alkylation damage in DNA involves Mag1 but not Tpa1. We observed that yeast strains without tpa1 are no more sensitive to MMS than WT yeast, whereas mag1-deficient yeast are ∼500-fold more sensitive to MMS. We therefore investigated the substrate specificity of Mag1 and found that it excises alkylated bases that are known AlkB substrates. In contrast, purified recombinant Tpa1 did not repair these alkylated DNA substrates, but it did exhibit the prolyl hydroxylase activity that has also been ascribed to it. A comparison of several of the kinetic parameters of Mag1 and its E. coli homolog AlkA revealed that Mag1 catalyzes base excision from known AlkB substrates with greater efficiency than does AlkA, consistent with an expanded role of yeast Mag1 in repair of alkylation damage. Our results challenge the proposal that Tpa1 directly functions in DNA repair and suggest that Mag1-initiated base excision repair compensates for the absence of oxidative dealkylation of alkylated nucleobases in budding yeast. This expanded role of Mag1, as compared with alkylation repair glycosylases in other organisms, could explain the extreme sensitivity of Mag1-deficient S. cerevisiae toward alkylation damage.

Distinguishing Specific and Nonspecific Complexes of Alkyladenine DNA Glycosylase.

Human alkyladenine DNA glycosylase (AAG) recognizes many alkylated and deaminated purine lesions and excises them to initiate the base excision DNA repair pathway. AAG employs facilitated diffusion to rapidly scan nonspecific sites and locate rare sites of damage. Nonspecific DNA binding interactions are critical to the efficiency of this search for damage, but little is known about the binding footprint or the affinity of AAG for nonspecific sites. We used biochemical and biophysical approaches to characterize the binding of AAG to both undamaged and damaged DNA. Although fluorescence anisotropy is routinely used to study DNA binding, we found unexpected complexities in the data for binding of AAG to DNA. Systematic comparison of different fluorescent labels and different lengths of DNA allowed binding models to be distinguished and demonstrated that AAG can bind with high affinity and high density to nonspecific DNA. Fluorescein-labeled DNA gave the most complex behavior but also showed the greatest potential to distinguish specific and nonspecific binding modes. We suggest a unified model that is expected to apply to many DNA binding proteins that exhibit affinity for nonspecific DNA. Although AAG strongly prefers to excise lesions from duplex DNA, nonspecific binding is comparable for single- and double-stranded nonspecific sites. The electrostatically driven binding of AAG to small DNA sites (∼5 nucleotides of single-stranded and ∼6 base pairs of duplex) facilitates the search for DNA damage in chromosomal DNA, which is bound by nucleosomes and other proteins.

Search for DNA damage by human alkyladenine DNA glycosylase involves early intercalation by an aromatic residue.

DNA repair enzymes recognize and remove damaged bases that are embedded in the duplex. To gain access, most enzymes use nucleotide flipping, whereby the target nucleotide is rotated 180° into the active site. In human alkyladenine DNA glycosylase (AAG), the enzyme that initiates base excision repair of alkylated bases, the flipped-out nucleotide is stabilized by intercalation of the side chain of tyrosine 162 that replaces the lesion nucleobase. Previous kinetic studies provided evidence for the formation of a transient complex that precedes the stable flipped-out complex, but it is not clear how this complex differs from nonspecific complexes. We used site-directed mutagenesis and transient-kinetic approaches to investigate the timing of Tyr162 intercalation for AAG. The tryptophan substitution (Y162W) appeared to be conservative, because the mutant protein retained a highly favorable equilibrium constant for flipping the 1,N6-ethenoadenine (ϵA) lesion, and the rate of N-glycosidic bond cleavage was identical to that of the wild-type enzyme. We assigned the tryptophan fluorescence signal from Y162W by removing two native tryptophan residues (W270A/W284A). Stopped-flow experiments then demonstrated that the change in tryptophan fluorescence of the Y162W mutant is extremely rapid upon binding to either damaged or undamaged DNA, much faster than the lesion-recognition and nucleotide flipping steps that were independently determined by monitoring the ϵA fluorescence. These observations suggest that intercalation by this aromatic residue is one of the earliest steps in the search for DNA damage and that this interaction is important for the progression of AAG from nonspecific searching to specific-recognition complexes.

Kinetic analyses of single-stranded break repair by human DNA ligase III isoforms reveal biochemical differences from DNA ligase I.

Humans have three genes encoding DNA ligases with conserved structural features and activities, but they also have notable differences. The LIG3 gene encodes a ubiquitous isoform in all tissues (LIG3α) and a germ line-specific splicing isoform (LIG3ß) that differs in the C-terminal domain. Both isoforms are found in the nucleus and the mitochondria. Here, we determined the kinetics and thermodynamics of single-stranded break ligation by LIG3α and LIG3ß and compared this framework to that of LIG1, the nuclear replicative ligase. The kinetic parameters of the LIG3 isoforms are nearly identical under all tested conditions, indicating that the BRCA1 C terminal (BRCT) domain specific to LIG3α does not alter ligation kinetics. Although LIG3 is only 22% identical to LIG1 across their conserved domains, the two enzymes had very similar maximal ligation rates. Comparison of the rate and equilibrium constants for LIG3 and LIG1 nevertheless revealed important differences. The LIG3 isoforms were seven times more efficient than LIG1 at ligating nicked DNA under optimal conditions, mainly because of their lower Km value for the DNA substrate. This could explain why LIG3 is less prone to abortive ligation than LIG1. Surprisingly, the affinity of LIG3 for Mg2+ was ten times weaker than that of LIG1, suggesting that Mg2+ availability regulates DNA ligation in vivo, because Mg2+ levels are higher in the mitochondria than in the nucleus. The biochemical differences between the LIG3 isoforms and LIG1 identified here will guide the understanding of both unique and overlapping biological roles of these critical enzymes.

Reactivity and Cross-Linking of 5'-Terminal Abasic Sites within DNA.

Nicking of the DNA strand immediately upstream of an internal abasic (AP) site produces 5'-terminal abasic (dRp) DNA. Both the intact and the nicked abasic species are reactive intermediates along the DNA base excision repair (BER) pathway and can be derailed by side reactions. Aberrant accumulation of the 5'-terminal abasic intermediate has been proposed to lead to cell death, so we explored its reactivity and compared it to the reactivity of the better-characterized internal abasic intermediate. We find that the 5'-terminal abasic group cross-links with the exocyclic amine of a nucleotide on the opposing strand to form an interstrand DNA-DNA cross-link (ICL). This cross-linking reaction has the same kinetic constants and follows the same pH dependence as the corresponding cross-linking reaction of intact abasic DNA, despite the changes in charge and flexibility engendered by the nick. However, the ICL that traps nicked abasic DNA has a shorter lifetime at physiological pH than the otherwise analogous ICL of intact abasic DNA due to the reversibility of the cross-linking reaction coupled with faster breakdown of the 5'-terminal abasic species via ß-elimination. Unlike internal abasic DNA, 5'-terminal abasic DNA can also react with exocyclic amines of unpaired nucleotides at the 3'-end of the nick, thereby bridging the nick by connecting DNA strands of the same orientation. The discovery and characterization of cross-links between 5'-terminal abasic sites and exocyclic amines of both opposing and adjacent nucleotides add to our knowledge of DNA damage with the potential to disrupt DNA transactions.

Transient Hoogsteen base pairs in canonical duplex DNA.

Sequence-directed variations in the canonical DNA double helix structure that retain Watson-Crick base-pairing have important roles in DNA recognition, topology and nucleosome positioning. By using nuclear magnetic resonance relaxation dispersion spectroscopy in concert with steered molecular dynamics simulations, we have observed transient sequence-specific excursions away from Watson-Crick base-pairing at CA and TA steps inside canonical duplex DNA towards low-populated and short-lived A•T and G•C Hoogsteen base pairs. The observation of Hoogsteen base pairs in DNA duplexes specifically bound to transcription factors and in damaged DNA sites implies that the DNA double helix intrinsically codes for excited state Hoogsteen base pairs as a means of expanding its structural complexity beyond that which can be achieved based on Watson-Crick base-pairing. The methods presented here provide a new route for characterizing transient low-populated nucleic acid structures, which we predict will be abundant in the genome and constitute a second transient layer of the genetic code.

Critical role of DNA intercalation in enzyme-catalyzed nucleotide flipping.

Nucleotide flipping is a common feature of DNA-modifying enzymes that allows access to target sites within duplex DNA. Structural studies have identified many intercalating amino acid side chains in a wide variety of enzymes, but the functional contribution of these intercalating residues is poorly understood. We used site-directed mutagenesis and transient kinetic approaches to dissect the energetic contribution of intercalation for human alkyladenine DNA glycosylase, an enzyme that initiates repair of alkylation damage. When AAG flips out a damaged nucleotide, the void in the duplex is filled by a conserved tyrosine (Y162). We find that tyrosine intercalation confers 140-fold stabilization of the extrahelical specific recognition complex, and that Y162 functions as a plug to slow the rate of unflipping by 6000-fold relative to the Y162A mutant. Surprisingly, mutation to the smaller alanine side chain increases the rate of nucleotide flipping by 50-fold relative to the wild-type enzyme. This provides evidence against the popular model that DNA intercalation accelerates nucleotide flipping. In the case of AAG, DNA intercalation contributes to the specific binding of a damaged nucleotide, but this enhanced specificity comes at the cost of reduced speed of nucleotide flipping.

Probing the DNA structural requirements for facilitated diffusion.

DNA glycosylases perform a genome-wide search to locate damaged nucleotides among a great excess of undamaged nucleotides. Many glycosylases are capable of facilitated diffusion, whereby multiple sites along the DNA are sampled during a single binding encounter. Electrostatic interactions between positively charged amino acids and the negatively charged phosphate backbone are crucial for facilitated diffusion, but the extent to which diffusing proteins rely on the double-helical structure DNA is not known. Kinetic assays were used to probe the DNA searching mechanism of human alkyladenine DNA glycosylase (AAG) and to test the extent to which diffusion requires B-form duplex DNA. Although AAG excises εA lesions from single-stranded DNA, it is not processive on single-stranded DNA because dissociation is faster than N-glycosidic bond cleavage. However, the AAG complex with single-stranded DNA is sufficiently stable to allow for DNA annealing when a complementary strand is added. This observation provides evidence of nonspecific association of AAG with single-stranded DNA. Single-strand gaps, bubbles, and bent structures do not impede the search by AAG. Instead, these flexible or bent structures lead to the capture of a nearby site of damage that is more efficient than that of a continuous B-form duplex. The ability of AAG to negotiate these helix discontinuities is inconsistent with a sliding mode of diffusion but can be readily explained by a hopping mode that involves microscopic dissociation and reassociation. These experiments provide evidence of relatively long-range hops that allow a searching protein to navigate around DNA binding proteins that would serve as obstacles to a sliding protein.

Kinetic mechanism for the flipping and excision of 1,N(6)-ethenoadenine by AlkA.

Escherichia coli 3-methyladenine DNA glycosylase II (AlkA), an adaptive response glycosylase with a broad substrate range, initiates base excision repair by flipping a lesion out of the DNA duplex and hydrolyzing the N-glycosidic bond. We used transient and steady state kinetics to determine the minimal mechanism for recognition and excision of 1,N(6)-ethenoadenine (εA) by AlkA. The natural fluorescence of this endogenously produced lesion allowed us to directly monitor the nucleotide flipping step. We found that AlkA rapidly and reversibly binds and flips out εA prior to N-glycosidic bond hydrolysis, which is the rate-limiting step of the reaction. The binding affinity of AlkA for the εA-DNA lesion is only 40-fold tighter than for a nonspecific site and 20-fold weaker than for the abasic DNA site. The mechanism of AlkA-catalyzed excision of εA was compared to that of the human alkyladenine DNA glycosylase (AAG), an independently evolved glycosylase that recognizes many of the same substrates. AlkA and AAG both catalyze N-glycosidic bond hydrolysis to release εA, and their overall rates of reaction are within 2-fold of each other. Nevertheless, we find dramatic differences in the kinetics and thermodynamics for binding to εA-DNA. AlkA catalyzes nucleotide flipping an order of magnitude faster than AAG; however, the equilibrium for flipping is almost 3 orders of magnitude more favorable for AAG than for AlkA. These results illustrate how enzymes that perform the same chemistry can use different substrate recognition strategies to effectively repair DNA damage.

Base excision repair enzymes protect abasic sites in duplex DNA from interstrand cross-links.

Hydrolysis of the N-glycosyl bond between a nucleobase and deoxyribose leaves an abasic site within duplex DNA. The abasic site can react with exocyclic amines of nucleobases on the complementary strand to form interstrand DNA-DNA cross-links (ICLs). We find that several enzymes from the base excision repair (BER) pathway protect an abasic site on one strand of a DNA duplex from cross-linking with an amine on the opposing strand. Human alkyladenine DNA glycosylase (AAG) and Escherichia coli 3-methyladenine DNA glycosylase II (AlkA) accomplish this by binding tightly to the abasic site and sequestering it. AAG protects an abasic site opposite T, the product of its canonical glycosylase reaction, by a factor of ∼10-fold, as estimated from its inhibition of the reaction of an exogenous amine with the damaged DNA. Human apurinic/apyrimidinic site endonuclease 1 and E. coli endonuclease III both decrease the amount of ICL at equilibrium by generating a single-strand DNA nick at the abasic position as it is liberated from the cross-link. The reversibility of the reaction between amines and abasic sites allows BER enzymes to counter the potentially disruptive effects of this type of cross-link on DNA transactions.

Isolating contributions from intersegmental transfer to DNA searching by alkyladenine DNA glycosylase.

Large genomes pose a challenge to DNA repair pathways because rare sites of damage must be efficiently located from among a vast excess of undamaged sites. Human alkyladenine DNA glycosylase (AAG) employs nonspecific DNA binding interactions and facilitated diffusion to conduct a highly redundant search of adjacent sites. This ensures that every site is searched, but could be a detriment if the protein is trapped in a local segment of DNA. Intersegmental transfer between DNA segments that are transiently in close proximity provides an elegant solution that balances global and local searching processes. It has been difficult to detect intersegmental transfer experimentally; therefore, we developed biochemical assays that allowed us to observe and measure the rates of intersegmental transfer by AAG. AAG has a flexible amino terminus that tunes its affinity for nonspecific DNA, but we find that it is not required for intersegmental transfer. As AAG has only a single DNA binding site, this argues against the bridging model for intersegmental transfer. The rates of intersegmental transfer are strongly dependent on the salt concentration, supporting a jumping mechanism that involves microscopic dissociation and capture by a proximal DNA site. As many DNA-binding proteins have only a single binding site, jumping may be a common mechanism for intersegmental transfer.

Defining the functional footprint for recognition and repair of deaminated DNA.

Spontaneous deamination of DNA is mutagenic, if it is not repaired by the base excision repair (BER) pathway. Crystallographic data suggest that each BER enzyme has a compact DNA binding site. However, these structures lack information about poorly ordered termini, and the energetic contributions of specific protein-DNA contacts cannot be inferred. Furthermore, these structures do not reveal how DNA repair intermediates are passed between enzyme active sites. We used a functional footprinting approach to define the binding sites of the first two enzymes of the human BER pathway for the repair of deaminated purines, alkyladenine DNA glycosylase (AAG) and AP endonuclease (APE1). Although the functional footprint for full-length AAG is explained by crystal structures of truncated AAG, the footprint for full-length APE1 indicates a much larger binding site than is observed in crystal structures. AAG turnover is stimulated in the presence of APE1, indicating rapid exchange of AAG and APE1 at the abasic site produced by the AAG reaction. The coordinated reaction does not require an extended footprint, suggesting that each enzyme engages the site independently. Functional footprinting provides unique information relative to traditional footprinting approaches and is generally applicable to any DNA modifying enzyme or system of enzymes.

An Open-Label Phase I Study of Metformin and Nelfinavir in Combination With Bortezomib in Patients With Relapsed and Refractory Multiple Myeloma.

BACKGROUND: In preclinical models, combining a GLUT4 inhibitor with an oxidative phosphorylation inhibitor shows synergistic therapeutic potential against multiple myeloma (MM). Thus, this study evaluated the safety and tolerability of repurposing metformin, a complex I inhibitor, and nelfinavir, a GLUT4 inhibitor, in combination with bortezomib for the treatment of relapsed/refractory MM that had progressed on all standard of care therapies. MATERIALS AND METHODS: This trial utilized a 3 + 3 dose escalation design with 3 dose levels planned for up to a maximum of 6 (21-day) cycles. Metformin and nelfinavir were administered for 14 of 21 days, and subQ bortezomib was administered to a portion of patients on days 1, 8, and 15. The primary objective was to determine the maximal tolerated dose, and the secondary objective was to evaluate the safety and overall response rate (ORR) of this combination. RESULTS: Nine patients were accrued with a median age of 65 (range: 42-81) and received a median of 7 prior lines of therapy (Range: 5-12). The first 3 patients received only metformin (500 mg BID) and nelfinavir (1250 mg BID) at the first dose level, with 1 patient experiencing an unconfirmed minimal response (MR) in the first cycle, 1 experiencing progressive disease after 1 cycle of treatment and 1 patient going off treatment prior to assessing response but with signs of progressive disease. Given the limited therapeutic activity, the upfront addition of bortezomib (1.3 mg/m2) was utilized for the subsequent 6 patients accrued. Three of these 6 patients went off study due to progressive disease, 1 patient achieved an unconfirmed partial response after 1 cycle of treatment but reported progressive disease in the subsequent cycle, 1 patient went off study to enter hospice, and the remaining patient experienced stable disease (SD) after receiving 6 cycles of clinical trial treatment. The study was closed before accrual to the next dose level was started. CONCLUSION: This is the first study to evaluate the safety and efficacy of this repurposed drug combination in this very difficult-to-treat population of relapsed and refractory MM. This was an overall negative study with no ORR observed. Fortunately, 1 patient experienced an SD response, allowing this combination to stabilize their disease until another novel therapy on a clinical trial was available.

DNA-N-glycosylases process novel O-glycosidic sites in DNA.

After the hydrolysis of the N-glycosyl bond between a damaged base and C1' of a deoxyribosyl moiety of DNA, human alkyladenine DNA glycosylase (AAG) and Escherichia coli 3-methyladenine DNA glycosylase II (AlkA) bind tightly to their abasic DNA products, potentially protecting these reactive species. Here we show that both AAG and AlkA catalyze reactions between bound abasic DNA and small, primary alcohols to form novel DNA-O-glycosides. The synthesis reactions are reversible, as the DNA-O-glycosides are converted back into abasic DNA upon being incubated with AAG or AlkA in the absence of alcohol. AAG and AlkA are therefore able to hydrolyze O-glycosidic bonds in addition to N-glycosyl bonds. The newly discovered DNA-O-glycosidase activities of both enzymes compare favorably with their known DNA-N-glycosylase activities: AAG removes both methanol and 1,N(6)-ethenoadenine (εA) from DNA with single-turnover rate constants that are 2.9 × 10(5)-fold greater than the corresponding uncatalyzed rates, whereas the rate enhancement of 3.7 × 10(7) for removal of methanol from DNA by AlkA is 300-fold greater than its rate enhancement for removal of εA from DNA. Although the biological significance of the DNA-O-glycosidase reactions is not known, the evolution of new DNA repair pathways may be aided by enzymes that practice catalytic promiscuity, such as these two unrelated DNA glycosylases.

The role of continental subduction in mantle metasomatism and carbon recycling revealed by melt inclusions in UHP eclogites.

Subduction is the main process that recycles surface material into the mantle. Fluids and melts derived by dehydration and partial melting reactions of subducted continental crust, a major reservoir of volatiles (i.e., H2O and CO2) and incompatible elements, can substantially metasomatize and refertilize the mantle. Here, we investigate glassy inclusions of silicate melt of continental origin found in Variscan ultrahigh-pressure eclogites to assess the continental crust contribution to mantle metasomatism and the journey of volatiles, carbon in particular, to the deep roots of mountain belts. We argue that the melt preserved in these inclusions is the agent responsible for mantle metasomatism and subsequent ultrapotassic magmatism in the Variscides. We propose that continental subduction can redistribute a substantial volume of carbon in the continental lithosphere, which is subsequently transferred to the continental crust during postcollisional magmatism and stored for a time length longer than that of the modern carbon cycle.