Search

The nuclear and subnuclear compartmentalization of the telomerase-associated protein and H/ACA ribonucleoprotein component dyskerin is an important although incompletely understood aspect of H/ACA ribonucleoprotein function. Four SUMOylation sites were previously identified in the C-terminal nuclear/nucleolar localization signal (N/NoLS) of dyskerin. We found that a cytoplasmic localized C-terminal truncation variant of dyskerin lacking most of the C-terminal N/NoLS represents an under-SUMOylated variant of dyskerin compared to wild-type dyskerin. We demonstrate that mimicking constitutive SUMOylation of dyskerin using a SUMO3 fusion construct can drive nuclear accumulation of this variant and that the SUMO site K467 in this N/NoLS is particularly important for the subnuclear localization of dyskerin to the nucleolus in a mature H/ACA complex assembly- and SUMO-dependent manner. We also characterize a novel SUMO-interacting motif in the mature H/ACA complex component GAR1 that mediates the interaction between dyskerin and GAR1. Mislocalization of dyskerin, either in the cytoplasm or excluded from the nucleolus, disrupts dyskerin function and leads to reduced interaction of dyskerin with the telomerase RNA. These data indicate a role for dyskerin C-terminal N/NoLS SUMOylation in regulating the nuclear and subnuclear localization of dyskerin, which is essential for dyskerin function as both a telomerase-associated protein and as an H/ACA ribonucleoprotein.

International audience; SUMOylation is a reversible post-translational modification that regulates several cellular processes including protein stability, subcellular localization, protein–protein interactions and plays a key role in the interferon (IFN) pathway and antiviral defense. In human, three ubiquitously expressed SUMO paralogs (SUMO1, 2 and 3) have been described for their implication in both intrinsic and innate immunity. Differential effects between SUMO paralogs are emerging such as their distinctive regulations of IFN synthesis, of IFN signaling and of the expression and function of IFN-stimulated gene (ISG) products. Several restriction factors are conjugated to SUMO and their modifications are further enhanced in response to IFN. Also, IFN itself was shown to increase global cellular SUMOylation and requires the presence of the E3 SUMO ligase PML that coordinates the assembly of PML nuclear bodies. This review focuses on differential effects of SUMO paralogs on IFN signaling and the stabilization/destabilization of ISG products, highlighting the crosstalk between SUMOylation and other post-translational modifications such as ubiquitination and ISGylation.

SummaryDyskerin, a telomerase-associated protein and H/ACA ribonucleoprotein complex component plays an essential role in human telomerase assembly and activity. The nuclear and subnuclear compartmentalization of dyskerin and the H/ACA complex is an important though incompletely understood aspect of H/ACA ribonucleoprotein function. The posttranslational modification, SUMOylation, targets a wide variety of proteins, including numerous RNA-binding proteins, and most identified targets reported to date localize to the nucleus. Four SUMOylation sites were previously identified in the C-terminal Nuclear/Nucleolar Localization Signal (N/NoLS) of dyskerin, each located within one of two lysine-rich clusters. We found that a cytoplasmic localized C-terminal truncation variant of dyskerin lacking most of the C-terminal N/NoLS and both lysine-rich clusters represents an under-SUMOylated variant of dyskerin compared to wildtype dyskerin. We demonstrate that mimicking constitutive SUMOylation of dyskerin using a SUMO3-fusion construct can drive nuclear accumulation of this variant, and that the SUMO site K467 in this N/NoLS is particularly important for the subnuclear localization of dyskerin to the nucleolus in a mature H/ACA complex assembly- and SUMO-dependent manner. We also characterize a novel SUMO-interacting motif in the mature H/ACA complex component GAR1 that mediates the interaction between dyskerin and GAR1. Mislocalization of dyskerin, either in the cytoplasm or excluded from the nucleolus, disrupts dyskerin function and leads to reduced interaction of dyskerin with the telomerase RNA. These data indicate a role for dyskerin C-terminal N/NoLS SUMOylation in regulating the nuclear and subnuclear localization of dyskerin, which is essential for dyskerin function as both a telomerase-associated protein and as an H/ACA ribonucleoprotein involved in rRNA and snRNA biogenesis.

The mechanistic target of rapamycin (mTOR) signaling is influenced by multiple regulatory proteins and post-translational modifications, however, underlying mechanisms remains unclear. Here, we report a novel role of small ubiquitin-like modifier (SUMO) in mTOR complex assembly and activity. By investigating the SUMOylation status of core mTOR components, we observed that the regulatory subunit, GβL, is modified by SUMO1, 2, and 3 isoforms. Using mutagenesis and mass spectrometry, we identified that GβL is SUMOylated at lysine sites K86, K215, K245, K261 and K305. We found that SUMO depletion reduces mTOR-Raptor and mTOR-Rictor complex formation and diminishes nutrient-induced mTOR signaling. Furthermore, we found that reconstitution with WT GβL but not SUMOylation defective KR mutant GβL promote mTOR signaling in GβL-depleted cells. Taken together, we report for the very first time that SUMO modifies GβL, influences the assembly of mTOR protein complexes, and regulates mTOR activity.

Rhes (Ras homolog enriched in the striatum) is a multifunctional protein that orchestrates striatal toxicity, motor behaviors and abnormal movements associated with dopaminergic signaling, Huntington disease and Parkinson disease signaling in the striatum. Rhes engineers membranous tunneling nanotube-like structures and promotes intercellular protein and cargoes transport. Recent study revealed Rhes also regulates mitophagy via the Nix receptor. Despite these studies, the mechanisms through which Rhes mediates these diverse functions remains unclear. Rhes belongs to a small GTPase family member and consists of a unique C-terminal Small Ubiquitin-like Modifier (SUMO) E3-like domain that promotes the post-translational modification (PTM) of proteins with SUMO (SUMOylation) by promoting “cross-SUMOylation” of SUMO enzymes SUMO E1 (Aos1/Uba2) and SUMO E2 ligase (Ubc-9). However, the identity of the SUMO substrates of Rhes remains largely unknown. By combining high throughput interactome and SUMO proteomics we report that Rhes regulates the SUMOylation of nuclear proteins that are involved in the regulation of gene transcription. While Rhes has increased the SUMOylation of histone deacetylase 1 (HDAC1) and histone 2B, it had decreased the SUMOylation of heterogeneous nuclear ribonucleoprotein M (HNRNPM), protein polybromo-1 (PBRM1) and E3 SUMO-protein ligase (PIASy). We also found that Rhes itself is SUMOylated at 5 different lysine residues (K32, K110, K114, K120, K124 and K245). Furthermore, we found that Rhes regulates the expression of genes involved in cellular morphogenesis and differentiation in the striatum, in a SUMO-dependent manner. Our findings thus provide a previously undescribed role for Rhes in regulating SUMOylation of nuclear targets and in orchestrating striatal gene expression via the SUMOylation.

Arsenic trioxide (ATO) is a therapeutic agent used to treat acute promyelocytic leukemia (APL), a disease caused by a chromosomal translocation of the retinoic acid receptor α (RARα) gene that can occur reciprocally with the promyelocytic leukemia (PML) gene. The mechanisms through which ATO exerts its effects on cells are not fully characterized though they involve the SUMOylation, the ubiquitylation, and the degradation of the PML/RARα oncoprotein through the PML moiety. To better understand the mechanisms that underlie the cytotoxicity induced with increasing ATO levels, we profiled the changes in protein SUMOylation, phosphorylation, and ubiquitylation on HEK293 cells following exposure to low (1 μM) or elevated (10 μM) ATO for 4 h. Our analyses revealed that a low dose of ATO resulted in the differential modification of selected substrates including the SUMOylation (K380, K394, K490, and K497) and ubiquitylation (K337, K401) of PML. These experiments also highlighted a number of unexpected SUMOylated substrates involved in DNA damage response (e.g., PCNA, YY1, and poly[ADP-ribose] polymerase 1 (PARP1)) and messenger RNA (mRNA) splicing (e.g., ACIN1, USP39, and SART1) that were regulated at higher ATO concentrations. Interestingly, additional enzymatic assays revealed that SUMOylation of PARP1 impeded its proteolytic cleavage by caspase-3, suggesting that SUMOylation could have a protective role in delaying cell apoptosis.

The depth of proteomic analyses is often limited by the overwhelming proportion of confounding background ions that compromise the identification and quantification of low abundance peptides. To alleviate these limitations, we present a new high field asymmetric waveform ion mobility spectrometry (FAIMS) interface that can be coupled to the Orbitrap Tribrid mass spectrometers. The interface provides several advantages over previous generations of FAIMS devices, including ease of operation, robustness, and high ion transmission. Replicate LC-FAIMS-MS/MS analyses (n = 100) of HEK293 protein digests showed stable ion current over extended time periods with uniform peptide identification on more than 10,000 distinct peptides. For complex tryptic digest analyses, the coupling of FAIMS to LC-MS/MS enabled a 30% gain in unique peptide identification compared with non-FAIMS experiments. Improvement in sensitivity facilitated the identification of low abundance peptides, and extended the limit of detection by almost an order of magnitude. The reduction in chimeric MS/MS spectra using FAIMS also improved the precision and the number of quantifiable peptides when using isobaric labeling with tandem mass tag (TMT) 10-plex reagent. We compared quantitative proteomic measurements for LC-MS/MS analyses performed using synchronous precursor selection (SPS) and LC-FAIMS-MS/MS to profile the temporal changes in protein abundance of HEK293 cells following heat shock for periods up to 9 h. FAIMS provided 2.5-fold increase in the number of quantifiable peptides compared with non-FAIMS experiments (30,848 peptides from 2,646 proteins for FAIMS versus 12,400 peptides from 1,229 proteins with SPS). Altogether, the enhancement in ion transmission and duty cycle of the new FAIMS interface extended the depth and comprehensiveness of proteomic analyses and improved the precision of quantitative measurements.

The Protein Inhibitor of Activated STAT 1 (PIAS1) is an E3 SUMO ligase that plays important roles in various cellular pathways, including STAT signaling, p53 pathway, and the steroid hormone signaling pathway. PIAS1 can SUMOylate PML (at Lys-65 and Lys-160) and PML-RARα promoting their ubiquitin-mediated degradation. Increasing evidence shows that PIAS1 is overexpressed in various human malignancies, including prostate and lung cancers. To understand the mechanism of action of PIAS1, we developed a quantitative SUMO proteomic approach to identify potential substrates of PIAS1 in a system-wide manner. Our analyses enabled the profiling of 983 SUMO sites on 544 proteins, of which 204 SUMO sites on 123 proteins were identified as putative PIAS1 substrates. These substrates are involved in different cellular processes, such as transcriptional regulation, DNA binding and cytoskeleton dynamics. Further functional studies on Vimentin (VIM), a type III intermediate filament protein involved in cytoskeleton organization and cell motility, revealed that PIAS1 exerts its effects on cell migration and cell invasion through the SUMOylation of VIM at Lys-439 and Lys-445 residues. VIM SUMOylation was necessary for its dynamic disassembly, and cells expressing a non-SUMOylatable VIM mutant showed reduced levels of proliferation and migration. Our approach not only provides a novel strategy for the identification of E3 SUMO ligase substrates, but also yields valuable biological insights into the unsuspected role of PIAS1 and VIM SUMOylation on cell motility.

Stable isotope labeling by amino acids in cell culture (SILAC) is routinely used to profile changes in protein and peptide abundance across different experimental paradigms. As with other quantitative proteomic approaches, the detection of peptide isotopomers can be limited by the presence of interference ions that ultimately affect the quality of quantitative measurements. Here, we evaluate high field asymmetric waveform ion mobility spectrometry (FAIMS) to improve the accuracy and dynamic range of quantitative proteomic analyses using SILAC. We compared quantitative measurements for tryptic digests of isotopically labeled protein extracts mixed in different ratios using LC-MS/MS with and without FAIMS. To further reduce sample complexity, we also examined the improvement in quantitative measurements when combining strong cation exchange (SCX) fractionation prior to LC-MS/MS analyses. Using the same amount of sample consumed, analyses performed using FAIMS provided more than 30% and 200% increase in the number of quantifiable peptides compared to LC-MS/MS performed with and without SCX fractionation, respectively. Furthermore, FAIMS reduced the occurrence of interfering isobaric ions and improved the accuracy of quantitative measurements. We leveraged the application of FAIMS in phosphoproteomic analyses to profile dynamic changes in protein phosphorylation in HEK293 cells subjected to heat shock for periods up to 20 min. In addition to the enhanced phosphoproteomic coverage, FAIMS also provided the ability to separate phosphopeptide isomers that often coelute and can be misassigned in conventional LC-MS/MS experiments.

Interferon (IFN) plays a central role in regulating host immune response to viral pathogens through the induction of IFN-Stimulated Genes (ISGs). IFN also enhances cellular SUMOylation and ISGylation, though the functional interplay between these modifications remains unclear. Here, we used a system-level approach to profile global changes in protein abundance in SUMO3-expressing cells stimulated by IFNα. These analyses revealed the stabilization of several ISG factors including SAMHD1, MxB, GBP1, GBP5, Tetherin/BST2 and members of IFITM, IFIT and IFI families. This process was correlated with enhanced IFNα-induced anti-HIV-1 and HSV-1 activities. Also IFNα upregulated protein ISGylation through increased abundance of E2 conjugating enzyme UBE2L6, and E3 ISG15 ligases TRIM25 and HERC5. Remarkably, TRIM25 depletion blocked SUMO3-dependent protein stabilization in response to IFNα. Our data identify a new mechanism by which SUMO3 regulates ISG product stability and reinforces the relevance of the SUMO pathway in controlling both the expression and functions of the restriction factors and IFN antiviral response.

Several regulators of SUMOylation have been previously linked to senescence but most targets of this modification in senescent cells remain unidentified. Using a two-step purification of a modified SUMO3, we profiled the SUMO proteome of senescent cells in a site-specific manner. We identified 25 SUMO sites on 23 proteins that were significantly regulated during senescence. Of note, most of these proteins were PML nuclear body (PML-NB) associated, which correlates with the increased number and size of PML-NBs observed in senescent cells. Interestingly, the sole SUMO E2 enzyme, UBC9, was more SUMOylated during senescence on its Lys-49. Functional studies of a UBC9 mutant at Lys-49 showed a decreased association to PML-NBs and the loss of UBC9’s ability to delay senescence. We thus propose both pro- and anti-senescence functions of protein SUMOylation.

We report that interferon (IFN) α treatment at short and long periods increases the global cellular SUMOylation and requires the presence of the SUMO E3 ligase promyelocytic leukemia protein (PML), the organizer of PML nuclear bodies (NBs). Several PML isoforms (PMLI-PMLVII) derived from a single PML gene by alternative splicing, share the same N-terminal region but differ in their C-terminal sequences. Introducing each of the human PML isoform in PML-negative cells revealed that enhanced SUMOylation in response to IFN is orchestrated by PMLIII and PMLIV. Large-scale proteomics experiments enabled the identification of 558 SUMO sites on 389 proteins, of which 172 sites showed differential regulation upon IFNα stimulation, including K49 from UBC9, the sole SUMO E2 protein. Furthermore, IFNα induces PML-dependent UBC9 transfer to the nuclear matrix where it colocalizes with PML within the NBs and enhances cellular SUMOylation levels. Our results demonstrate that SUMOylated UBC9 and PML are key players for IFN-increased cellular SUMOylation.

The small ubiquitin-like modifier (SUMO) is a member of the family of ubiquitin-like modifiers (UBLs) and is involved in important cellular processes, including DNA damage response, meiosis and cellular trafficking. The large-scale identification of SUMO peptides in a site-specific manner is challenging not only because of the low abundance and dynamic nature of this modification, but also due to the branched structure of the corresponding peptides that further complicate their identification using conventional search engines. Here, we exploited the unusual structure of SUMO peptides to facilitate their separation by high-field asymmetric waveform ion mobility spectrometry (FAIMS) and increase the coverage of SUMO proteome analysis. Upon trypsin digestion, branched peptides contain a SUMO remnant side chain and predominantly form triply protonated ions that facilitate their gas-phase separation using FAIMS. We evaluated the mobility characteristics of synthetic SUMO peptides and further demonstrated the application of FAIMS to profile the changes in protein SUMOylation of HEK293 cells following heat shock, a condition known to affect this modification. FAIMS typically provided a 10-fold improvement of detection limit of SUMO peptides, and enabled a 36% increase in SUMO proteome coverage compared to the same LC-MS/MS analyses performed without FAIMS. Graphical Abstract ᅟ.

Ubiquitin and ubiquitin-like modifiers (UBLs) such as small ubiquitin-like modifier (SUMO) can act as antagonists to one another by competing to occupy similar residues in the proteome. In addition, SUMO and ubiquitin can be coupled to each other at key lysine residues to form highly branched protein networks. The interplay between these modifications governs important biological processes such as double-strand break repair and meiotic recombination. We recently developed an approach that permits the identification of proteins that are modified by both SUMOylation and ubiquitylation. This protocol requires cells that express a mutant 6×His-SUMO3m protein that has had its C terminus modified from QQQTGG to RNQTGG, enabling the purification of SUMOylated peptides and their identification by tandem mass spectrometry (MS/MS). Cells are lysed under denaturing conditions, and the SUMOylated proteins are purified on nickel-nitrilotriacetic acid (Ni-NTA) resin via the 6×His on the SUMO3m construct. After on-bead digestion using trypsin, ubiquitylated peptides are enriched by immunoprecipitation, and the flow-through from this step is subjected to anti-SUMO immunoprecipitation. The SUMOylated peptides are fractionated on strong cation exchange (SCX) StageTips to enhance the coverage of the SUMO proteome. The ubiquitylated and SUMOylated peptides are analyzed separately by liquid chromatography (LC)-MS/MS and identified with MaxQuant. We demonstrate how this approach can be used to identify temporal changes in SUMOylated and ubiquitylated proteins in response to, for instance, heat shock and proteasome inhibition. The procedure requires 3 d when starting from cell pellets and yields >8,000 SUMO sites and >3,500 ubiquitin sites from 16 mg of cell extract.

DNA interstrand cross-links (ICL) are among the most cytotoxic lesions found in biological systems. O

O(6) -Alkylguanine-DNA alkyltransferases (AGTs) are responsible for the removal of O(6) -alkyl 2'-deoxyguanosine (dG) and O(4) -alkyl thymidine (dT) adducts from the genome. Unlike the E. coli OGT (O(6) -alkylguanine-DNA-alkyltransferase) protein, which can repair a range of O(4) -alkyl dT lesions, human AGT (hAGT) only removes methyl groups poorly. To uncover the influence of the C5 methyl group of dT on AGT repair, oligonucleotides containing O(4) -alkyl 2'-deoxyuridines (dU) were prepared. The ability of E. coli AGTs (Ada-C and OGT), human AGT, and an OGT/hAGT chimera to remove O(4) -methyl and larger adducts (4-hydroxybutyl and 7-hydroxyheptyl) from dU were examined and compared to those relating to the corresponding dT species. The absence of the C5 methyl group resulted in an increase in repair observed for the O(4) -methyl adducts by hAGT and the chimera. The chimera was proficient at repairing larger adducts at the O(4) atom of dU. There was no observed correlation between the binding affinities of the AGT homologues to adduct-containing oligonucleotides and the amounts of repair measured.

O(6)-alkylguanine-DNA alkyltransferases (AGT) are responsible for the removal of alkylation at both the O(6) atom of guanine and O(4) atom of thymine. AGT homologues show vast substrate differences with respect to the size of the adduct and which alkylated atoms they can restore. The human AGT (hAGT) has poor capabilities for removal of methylation at the O(4) atom of thymidine, which is not the case in most homologues. No structural data are available to explain this poor hAGT repair. We prepared and characterized O(6)G-butylene-O(4)T (XLGT4) and O(6)G-heptylene-O(4)T (XLGT7) interstrand cross-linked (ICL) DNA as probes for hAGT and the Escherichia coli homologues, OGT and Ada-C, for the formation of DNA-AGT covalent complexes. XLGT7 reacted only with hAGT and did so with a cross-linking efficiency of 25%, while XLGT4 was inert to all AGT tested. The hAGT mediated repair of XLGT7 occurred slowly, on the order of hours as opposed to the repair of O(6)-methyl-2'-deoxyguanosine which requires seconds. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the repair reaction revealed the formation of a covalent complex with an observed migration in accordance with a DNA-AGT complex. The identity of this covalent complex, as determined by mass spectrometry, was composed of a heptamethylene bridge between the O(4) atom of thymidine (in an 11-mer DNA strand) to residue Cys145 of hAGT. This procedure can be applied to produce well-defined covalent complexes between AGT with DNA.

Crosstalk between the SUMO and ubiquitin pathways has recently been reported. However, no approach currently exists to determine the interrelationship between these modifications. Here, we report an optimized immunoaffinity method that permits the study of both protein ubiquitylation and SUMOylation from a single sample. This method enables the unprecedented identification of 10,388 SUMO sites in HEK293 cells. The sequential use of SUMO and ubiquitin remnant immunoaffinity purification facilitates the dynamic profiling of SUMOylated and ubiquitylated proteins in HEK293 cells treated with the proteasome inhibitor MG132. Quantitative proteomic analyses reveals crosstalk between substrates that control protein degradation, and highlights co-regulation of SUMOylation and ubiquitylation levels on deubiquitinase enzymes and the SUMOylation of proteasome subunits. The SUMOylation of the proteasome affects its recruitment to promyelocytic leukemia protein (PML) nuclear bodies, and PML lacking the SUMO interacting motif fails to colocalize with SUMOylated proteasome further demonstrating that this motif is required for PML catabolism., Ubiquitylation and SUMOylation are two important related post-translational modifications. Here the authors present an approach for the simultaneous identification and quantification of protein-wide SUMO and ubiquitin sites from a single sample, uncovering widespread crosstalk between the two modifications.

Protein SUMOylation regulates the activity of a wide range of cellular substrates, and the identification of small ubiquitin-related modifier (SUMO)-modified sites is often required to understand how this modification affects protein function. However, the site-specific identification of modified lysine residues by mass spectrometry (MS) remains challenging because of the dynamic nature of this modification, its low stoichiometry and the relatively large SUMO remnant left on peptide backbones after tryptic digestion. Here we report a versatile method to identify sites and to profile the extent of modification on recombinant proteins from in vitro SUMOylation assays. We define the steps required for sample preparation, and we describe how to perform proper controls and conduct the liquid chromatography-MS (LC-MS) and bioinformatics analyses. Native protein substrates can be used for the assay, although we recommend the use of His-tagged proteins to facilitate removal of contaminants. The procedure was developed for human SUMO paralogs, and it requires

This protocol describes the preparation of O4-thymidine-alkylene-O4-thymidine dimer bis-phosphoramidites and precursors for incorporation into DNA sequences to produce site-specific DNA interstrand cross-links. Linkers are introduced at the 4-position of thymidine by reacting the sodium salt of a diol with a pyrimidinyl-convertible nucleoside to produce mono-adducts, which then undergo reaction with a stoichiometric equivalent of a pyrimidinyl-convertible nucleoside under basic conditions to form O4-thymidine-alkylene-O4-thymidine dimers. Bis-phosphoramidites are incorporated into oligonucleotides by solid-phase synthesis, and mild conditions for deprotection and cleavage from the solid support are employed to prevent degradation of the thymidine modifications. Purification of these cross-linked oligonucleotides is performed by denaturing polyacrylamide gel electrophoresis. This approach allows for the preparation of cross-linked DNA substrates in quantities and purity sufficient for a wide range of biophysical experiments and biochemical studies as substrates to investigate DNA repair pathways. Curr. Protoc. Nucleic Acid Chem. 55:5.13.1-5.13.19. © 2013 by John Wiley & Sons, Inc. Keywords: DNA interstrand cross-link; chemically modified oligonucleotide; oligonucleotide synthesis; solid-phase synthesis; DNA damage; DNA repair

O6-Alkylguanine-DNA alkyltransferases (AGTs) are proteins responsible for the removal of mutagenic alkyl adducts at the O6-atom of guanine and O4-atom of thymine. In the current study we set out to understand the role of the Ser134 residue in the Escherichia coli AGT variant OGT on substrate discrimination. The S134P mutation in OGT increased the ability of the protein to repair both O6-adducts of guanine and O4-adducts of thymine. However, the S134P variant was unable, like wild-type OGT, to repair an interstrand cross-link (ICL) bridging two O6-atoms of guanine in a DNA duplex. When compared to the human AGT protein (hAGT), the S134P OGT variant displayed reduced activity towards O6-alkylation but a much broader substrate range for O4-alkylation damage reversal. The role of residue 134 in OGT is similar to its function in the human homolog, where Pro140 is crucial in conferring on hAGT the capability to repair large adducts at the O6-position of guanine. Finally, a method to generate a covalent conjugate between hAGT and a model nucleoside using a single-stranded oligonucleotide substrate is demonstrated.

A soluble human O6-alkylguanine-DNA alkyltransferase (hAGT) chimera was engineered containing the active site of OGT (residues 139–159) and an additional S134P mutation. The resulting hAGT chimera not only retained hAGT's ability to repair bulky O6-alkylene-2′-deoxyguanosine interstrand cross-linked DNA damage but also displayed enhanced repair of various O4-alkyl thymidine adducts.

DNA duplexes containing a directly opposed O(4)-2'-deoxythymidine-alkyl-O(4)-2'-deoxythymidine (O(4)-dT-alkyl-O(4)-dT) interstrand cross-link (ICL) have been prepared by the synthesis of cross-linked nucleoside dimers which were converted to phosphoramidites to produce site specific ICL. ICL duplexes containing alkyl chains of four and seven methylene groups were prepared and characterized by mass spectrometry and nuclease digests. Thermal denaturation experiments revealed four and seven methylene containing ICL increased the T(m) of the duplex with respect to the non-cross-linked control with an observed decrease in enthalpy based on thermodynamic analysis of the denaturation curves. Circular dichroism experiments on the ICL duplexes indicated minimal difference from B-form DNA structure. These ICL were used for DNA repair studies with O(6)-alkylguanine DNA alkyltransferase (AGT) proteins from human (hAGT) and E. coli (Ada-C and OGT), whose purpose is to remove O(6)-alkylguanine and in some cases O(4)-alkylthymine lesions. It has been previously shown that hAGT can repair O(6)-2'-deoxyguanosine-alkyl-O(6)-2'-deoxyguanosine ICL. The O(4)-dT-alkyl-O(4)-dT ICL prepared in this study were found to evade repair by hAGT, OGT and Ada-C. Electromobility shift assay (EMSA) results indicated that the absence of any repair by hAGT was not a result of binding. OGT was the only AGT to show activity in the repair of oligonucleotides containing the mono-adducts O(4)-butyl-4-ol-2'-deoxythymidine and O(4)-heptyl-7-ol-2'-deoxythymidine. Binding experiments conducted with hAGT demonstrated that the protein bound O(4)-alkylthymine lesions with similar affinities to O(6)-methylguanine, which hAGT repairs efficiently, suggesting the lack of O(4)-alkylthymine repair by hAGT is not a function of recognition.

O6-2′-Deoxyguanosine-alkyl-O6-2′-deoxyguanosine interstrand DNA cross-links (ICLs) with a four and seven methylene linkage in a 5′-GNC- motif have been synthesized and their repair by human O6-alkylguanine-DNA alkyltransferase (hAGT) investigated. Duplexes containing 11 base-pairs with the ICLs in the center were assembled by automated DNA solid-phase synthesis using a cross-linked 2′-deoxyguanosine dimer phosphoramidite, prepared via a seven step synthesis which employed the Mitsunobu reaction to introduce the alkyl lesion at the O6 atom of guanine. Introduction of the four and seven carbon ICLs resulted in no change in duplex stability based on UV thermal denaturation experiments compared to a non-cross-linked control. Circular dichroism spectra of these ICL duplexes exhibited features of a B-form duplex, similar to the control, suggesting that these lesions induce little overall change in structure. The efficiency of repair by hAGT was examined and it was shown that hAGT repairs both ICL containing duplexes, with the heptyl ICL repaired more efficiently relative to the butyl cross-link. These results were reproducible with various hAGT mutants including one that contains a novel V148L mutation. The ICL duplexes displayed similar binding affinities to a C145S hAGT mutant compared to the unmodified duplex with the seven carbon containing ICLs displaying slightly higher binding. Experiments with CHO cells to investigate the sensitivity of these cells to busulfan and hepsulfam demonstrate that hAGT reduces the cytotoxicity of hepsulfam suggesting that the O6-2′-deoxyguanosine-alkyl-O6-2′-deoxyguanosine interstrand DNA cross-link may account for at least part of the cytotoxicity of this agent.