Enhanced binding of guanylated poly(A) RNA by the LaM domain of LARP1.

La-related proteins (LARPs) are a family of RNA-binding proteins that share a conserved La motif (LaM) domain. LARP1 plays a role in regulating ribosomal protein synthesis and stabilizing mRNAs and has a unique structure without an RNA binding RRM domain adjoining the LaM domain. In this study, we investigated the physical basis for LARP1 specificity for poly(A) sequences and observed an unexpected bias for sequences with single guanines. Multiple guanine substitutions did not increase the affinity, demonstrating preferential recognition of singly guanylated sequences. We also observed that the cyclic di-nucleotides in the cCAS/STING pathway, cyclic-di-GMP and 3',3'-cGAMP, bound with sub-micromolar affinity. Isothermal titration measurements were complemented by high-resolution crystal structures of the LARP1 LaM with six different RNA ligands, including two stereoisomers of a phosphorothioate linkage. The selectivity for singly substituted poly(A) sequences suggests LARP1 may play a role in the stabilizing effect of poly(A) tail guanylation. [Figure: see text].

Structural basis of 3'-end poly(A) RNA recognition by LARP1.

La-related proteins (LARPs) comprise a family of RNA-binding proteins involved in a wide range of posttranscriptional regulatory activities. LARPs share a unique tandem of two RNA-binding domains, La motif (LaM) and RNA recognition motif (RRM), together referred to as a La-module, but vary in member-specific regions. Prior structural studies of La-modules reveal they are pliable platforms for RNA recognition in diverse contexts. Here, we characterize the La-module of LARP1, which plays an important role in regulating synthesis of ribosomal proteins in response to mTOR signaling and mRNA stabilization. LARP1 has been well characterized functionally but no structural information exists for its La-module. We show that unlike other LARPs, the La-module in LARP1 does not contain an RRM domain. The LaM alone is sufficient for binding poly(A) RNA with submicromolar affinity and specificity. Multiple high-resolution crystal structures of the LARP1 LaM domain in complex with poly(A) show that it is highly specific for the RNA 3'-end, and identify LaM residues Q333, Y336 and F348 as the most critical for binding. Use of a quantitative mRNA stabilization assay and poly(A) tail-sequencing demonstrate functional relevance of LARP1 RNA binding in cells and provide novel insight into its poly(A) 3' protection activity.

Interdomain allostery promotes assembly of the poly(A) mRNA complex with PABP and eIF4G.

Many RNA-binding proteins contain multiple single-strand nucleic acid-binding domains and assemble into large multiprotein messenger ribonucleic acid protein (mRNP) complexes. The mechanisms underlying the self-assembly of these complexes are largely unknown. In eukaryotes, the association of the translation factors polyadenylate-binding protein-1 (PABP) and eIF4G is essential for high-level expression of polyadenylated mRNAs. Here, we report the crystal structure of the ternary complex poly(A)(11)·PABP(1-190)·eIF4G(178-203) at 2.0 Å resolution. Our NMR and crystallographic data show that eIF4G interacts with the RRM2 domain of PABP. Analysis of the interaction by small-angle X-ray scattering, isothermal titration calorimetry, and electromobility shift assays reveals that this interaction is allosterically regulated by poly(A) binding to PABP. Furthermore, we have confirmed the importance of poly(A) for the endogenous PABP and eIF4G interaction in immunoprecipitation experiments using HeLa cell extracts. Our findings reveal interdomain allostery as a mechanism for cooperative assembly of RNP complexes.

Influence of nucleotide modifications at the C2' position on the Hoogsteen base-paired parallel-stranded duplex of poly(A) RNA.

Polyadenylate (poly(A)) has the ability to form a parallel duplex with Hoogsteen adenine:adenine base pairs at low pH or in the presence of ammonium ions. In order to evaluate the potential of this structural motif for nucleic acid-based nanodevices, we characterized the effects on duplex stability of substitutions of the ribose sugar with 2'-deoxyribose, 2'-O-methyl-ribose, 2'-deoxy-2'-fluoro-ribose, arabinose and 2'-deoxy-2'-fluoro-arabinose. Deoxyribose substitutions destabilized the poly(A) duplex both at low pH and in the presence of ammonium ions: no duplex formation could be detected with poly(A) DNA oligomers. Other sugar C2' modifications gave a variety of effects. Arabinose and 2'-deoxy-2'-fluoro-arabinose nucleotides strongly destabilized poly(A) duplex formation. In contrast, 2'-O-methyl and 2'-deoxy-2'-fluoro-ribo modifications were stabilizing either at pH 4 or in the presence of ammonium ions. The differential effect suggests they could be used to design molecules selectively responsive to pH or ammonium ions. To understand the destabilization by deoxyribose, we determined the structures of poly(A) duplexes with a single DNA residue by nuclear magnetic resonance spectroscopy and X-ray crystallography. The structures revealed minor structural perturbations suggesting that the combination of sugar pucker propensity, hydrogen bonding, pKa shifts and changes in hydration determine duplex stability.

Structural basis of interstrand cross-link repair by O6-alkylguanine DNA alkyltransferase.

DNA interstrand cross-links (ICL) are among the most cytotoxic lesions found in biological systems. O6-Alkylguanine DNA alkyltransferases (AGTs) are capable of removing alkylation damage from the O6-atom of 2'-deoxyguanosine and the O4-atom of thymidine. Human AGT (hAGT) has demonstrated the ability to repair an interstrand cross-linked duplex where two O6-atoms of 2'-deoxyguanosine were tethered by a butylene (XLGG4) or heptylene (XLGG7) linkage. However, the analogous ICL between the O4-atoms of thymidine was found to evade repair. ICL duplexes connecting the O4-atoms of 2'-deoxyuridine by a butylene (XLUU4) or heptylene (XLUU7) linkage have been prepared to examine the influence of the C5-methyl group on AGT-mediated repair. Both XLUU4 and XLUU7 were refractory to repair by human and E. coli (OGT and Ada-C) AGTs with comparably low µM dissociation constants for 2 : 1 or 4 : 1 AGT/DNA stoichiometries. The solution structures of two heptylene linked DNA duplexes (CGAAAYTTTCG)2, XLUU7 (Y = dU) and XLGG7 (Y = dG), were solved and the global structures were virtually identical with a RMSD of 1.22 Å. The ICL was found to reside in the major groove for both duplexes. The linkage adopts an E conformation about the C4-O4 bond for XLUU7 whereas a Z conformation about the C6-O6 bond was observed for XLGG7. This E versus Z conformation may partially account for hAGTs discrimination towards the repair of these ICL, supported by the crystal structures of hAGT with various substrates which have been observed to adopt a Z conformation. In addition, a higher mobility at the ICL site for XLUU7 is observed relative to XLGG7 that may play a role in repair by hAGT. Taken together, these findings provide insights on the AGT-mediated repair of cytotoxic ICL in terms of its processing capability and substrate specificity.

Altering Residue 134 Confers an Increased Substrate Range of Alkylated Nucleosides to the E. coli OGT Protein.

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.

The solution structure of double helical arabino nucleic acids (ANA and 2'F-ANA): effect of arabinoses in duplex-hairpin interconversion.

We report here the first structure of double helical arabino nucleic acid (ANA), the C2'-stereoisomer of RNA, and the 2'-fluoro-ANA analogue (2'F-ANA). A chimeric dodecamer based on the Dickerson sequence, containing a contiguous central segment of arabino nucleotides, flanked by two 2'-deoxy-2'F-ANA wings was studied. Our data show that this chimeric oligonucleotide can adopt two different structures of comparable thermal stabilities. One structure is a monomeric hairpin in which the stem is formed by base paired 2'F-ANA nucleotides and the loop by unpaired ANA nucleotides. The second structure is a bimolecular duplex, with all the nucleotides (2'F-ANA and ANA) forming Watson-Crick base pairs. The duplex structure is canonical B-form, with all arabinoses adopting a pure C2'-endo conformation. In the ANA:ANA segment, steric interactions involving the 2'-OH substituent provoke slight changes in the glycosidic angles and, therefore, in the ANA:ANA base pair geometry. These distortions are not present in the 2'F-ANA:2'F-ANA regions of the duplex, where the -OH substituent is replaced by a smaller fluorine atom. 2'F-ANA nucleotides adopt the C2'-endo sugar pucker and fit very well into the geometry of B-form duplex, allowing for favourable 2'F···H8 interactions. This interaction shares many features of pseudo-hydrogen bonds previously observed in 2'F-ANA:RNA hybrids and in single 2'F-ANA nucleotides.

Preparation of covalently linked complexes between DNA and O(6)-alkylguanine-DNA alkyltransferase using interstrand cross-linked DNA.

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.

O4-alkyl-2'-deoxythymidine cross-linked DNA to probe recognition and repair by O6-alkylguanine DNA alkyltransferases.

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.

Cross-link structure affects replication-independent DNA interstrand cross-link repair in mammalian cells.

DNA interstrand cross-links (ICLs) are cytotoxic products of common anticancer drugs and cellular metabolic processes, whose mechanism(s) of repair remains poorly understood. In this study, we show that cross-link structure affects ICL repair in nonreplicating reporter plasmids that contain a mispaired N(4)C-ethyl-N(4)C (C-C), N3T-ethyl-N3T (T-T), or N1I-ethyl-N3T (I-T) ICL. The T-T and I-T cross-links obstruct the hydrogen bond face of the base and mimic the N1G-ethyl-N3C ICL created by bis-chloroethylnitrosourea, whereas the C-C cross-link does not interfere with base pair formation. Host-cell reactivation (HCR) assays in human and hamster cells showed that repair of these ICLs primarily involves the transcription-coupled nucleotide excision repair (TC-NER) pathway. Repair of the C-C ICL was 5-fold more efficient than repair of the T-T or I-T ICLs, suggesting the latter cross-links hinder lesion bypass following initial ICL unhooking. The level of luciferase expression from plasmids containing a C-C cross-link remnant on either the transcribed or nontranscribed strand increased in NER-deficient cells, indicating NER involvement occurs at a step prior to remnant removal, whereas expression from similar T-T remnant plasmids was inhibited in NER-deficient cells, demonstrating NER is required for remnant removal. Sequence analysis of repaired plasmids showed a high proportion of C residues inserted at the site of the T-T and I-T cross-links, and HCR assays showed that Rev1 was likely responsible for these insertions. In contrast, both C and G residues were inserted at the C-C cross-link site, and Rev1 was not required for repair, suggesting replicative or other translesion polymerases can bypass the C-C remnant.

Synthesis and characterization of an O(6)-2'-deoxyguanosine-alkyl-O(6)-2'-deoxyguanosine interstrand cross-link in a 5'-GNC motif and repair by human O(6)-alkylguanine-DNA alkyltransferase.

O(6)-2'-Deoxyguanosine-alkyl-O(6)-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 O(6) 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 O(6)-2'-deoxyguanosine-alkyl-O(6)-2'-deoxyguanosine interstrand DNA cross-link may account for at least part of the cytotoxicity of this agent.

Effect of cross-link structure on DNA interstrand cross-link repair synthesis.

DNA interstrand cross-links (ICLs) are products of chemotherapeutic agents and cellular metabolic processes that block both replication and transcription. If left unrepaired, ICLs are extremely toxic to cells, and ICL repair mechanisms contribute to the survival of certain chemotherapeutic resistance tumors. A critical step in ICL repair involves unhooking the cross-link. In the absence of a homologous donor sequence, the resulting gap can be filled in by a repair synthesis step involving bypass of the cross-link remnant. Here, we examine the effect of cross-link structure on the ability of unhooked DNA substrates to undergo repair synthesis in mammalian whole cell extracts. Using 32P incorporation assays, we found that repair synthesis occurs efficiently past the site of damage when a DNA substrate containing a single N4C-ethyl-N4C cross-link is incubated in HeLa or Chinese hamster ovary cell extracts. This lesion, which can base pair with deoxyguanosine, is readily bypassed by both Escherichia coli DNA polymerase I and T7 DNA polymerase in a primer extension assay. In contrast, bypass was not observed in the primer extension assay or in mammalian cell extracts when DNA substrates containing a N3T-ethyl-N3T or N1I-ethyl-N3T cross-link, whose linkers obstruct the hydrogen bond face of the bases, were used. A modified phosphorothioate sequencing method was used to analyze the ICL repair patches created in the mammalian cell extracts. In the case of the N4C-ethyl-N4C substrate, the repair patch spanned the site of the cross-link, and the lesion was bypassed in an error-free manner. However, although the N3T-ethyl-N3T and N1I-ethyl-N3T substrates were unhooked in the extracts, bypass was not detected. These and our previous results suggest that although the chemical structure of an ICL may not affect initial cross-link unhooking, it can play a significant role in subsequent processing of the cross-link. Understanding how the physical and chemical differences of ICLs affect repair may provide a better understanding of the cytotoxic and mutagenic potential of specific ICLs.

Distortion-dependent unhooking of interstrand cross-links in mammalian cell extracts.

Interstrand cross-links (ICLs) are formed by many chemotherapeutic agents and may also arise endogenously. The mechanisms used to repair these lesions remain unclear in mammalian cells. Repair in Escherichia coli and Saccharomyces cerevisiae requires an initial unhooking step to release the tethered DNA strands. We used a panel of linear substrates containing different site-specific ICLs to characterize how structure affects ICL processing in mammalian cell extracts. We demonstrate that ICL-induced distortions affect NER-dependent and -independent processing events. The NER-dependent pathway produces dual incisions 5' to the site of the ICL as described previously [Bessho, T., et al. (1997) Mol. Cell. Biol. 17 (12), 6822-6830] but does not release the cross-link. Surprisingly, we also found that the interstrand cross-linked duplexes were unhooked in mammalian cell extracts in a manner independent of the NER pathway. Unhooking occurred identically in extracts prepared from human and rodent cells and is dependent on ATP hydrolysis and metal ions. The structure of the unhooked product was characterized and was found to contain the remnant of the cross-link. Both the NER-mediated dual 5' incisions and unhooking reactions were greatly stimulated by ICL-induced distortions, including increased local flexibility and disruption of base pairs surrounding the site of the ICL. These results suggest that in DNA not undergoing transcription or replication, distortions induced by the presence of an ICL could contribute significantly to initial cross-link recognition and processing.

Repair of O6-G-alkyl-O6-G interstrand cross-links by human O6-alkylguanine-DNA alkyltransferase.

O (6)-Alkylguanine-DNA alkyltransferase (AGT) plays an important role by protecting cells from alkylating agents. This reduces the frequency of carcinogenesis and mutagenesis initiated by such agents, but AGT also provides a major resistance mechanism to some chemotherapeutic drugs. To improve our understanding of the AGT-mediated repair reaction and our understanding of the spectrum of repairable damage, we have studied the ability of AGT to repair interstrand cross-link DNA damage where the two DNA strands are joined via the guanine- O (6) in each strand. An oligodeoxyribonucleotide containing a heptane cross-link was repaired with initial formation of an AGT-oligo complex and further reaction of a second AGT molecule yielding a hAGT dimer and free oligo. However, an oligodeoxyribonucleotide with a butane cross-link was a very poor substrate for AGT-mediated repair, and only the first reaction that forms an AGT-oligo complex could be detected. Models of the reaction of these substrates in the AGT active site show that the DNA duplex is forced apart locally to repair the first guanine. This reaction is greatly hindered with the butane cross-link, which is mostly buried in the active site pocket and limited in conformational flexibility. This limitation also prevents the adoption of a conformation for the second reaction to repair the AGT-oligo complex. These results are consistent with the postulated mechanism of AGT repair that involves DNA binding and flipping of the substrate nucleotide and indicate that hAGT can repair some types of interstrand cross-link damage.

Synthesis and characterization of DNA duplexes containing an N3T-ethyl-N3T interstrand crosslink in opposite orientations.

DNA duplexes containing an ethyl interstrand crosslink that bridges the N3 atoms of thymidines on the opposite strands have been synthesized using an approach that combines conventional solid phase oligonucleotide synthesis and the selective removal of protecting groups of a crosslinked thymidine dimer. This approach allows for the assembly of a crosslinked duplex directly on the solid support. Duplexes that contain a N3T-ethyl-N3T interstrand crosslink in a staggered orientation at either a -TA- or -AT-step in a duplex have been prepared. When placed in an -AT- step of a duplex the effect was stabilizing relative to the non-crosslinked control duplex (deltaTm= +24 degrees C) and this crosslinked duplex was found to efficiently form multimers in the presence of T4 ligase. In the case of the -TA- crosslinked duplex the stabilizing effect was less pronounced (deltaT.= +6 degrees C) and likewise did not undergo self ligation under identical conditions. Molecular modeling studies suggested that the -AT- containing lesion had little deviation in structure relative to the non-crosslinked duplex DNA control, whereas the -TA- crosslinked duplex exhibited significant buckling of the base pairs flanking the lesion.

Preparation of interstrand cross-linked DNA oligonucleotide duplexes.

Reaction of cellular DNA with environmental and chemotherapeutic agents can give rise to a variety of lesions including interstrand cross-links. Because interstrand cross-links can prevent DNA strand separation and thus DNA transcription and replication, they represent a serious impediment to cell survival. Cells have developed mechanisms to repair interstrand cross-links in their DNA and in the case of tumor cells, this can lead to resistance to chemotherapeutic agents. Efforts to investigate the mechanisms by which interstrand cross-links are repaired have been hampered by the difficulty of preparing sufficient quantities of well characterized substrates for physical and biochemical studies. This review will describe synthetic strategies that have been developed to synthesize short DNA oligonucleotide duplexes that contain interstrand cross-links. These short duplexes can be used to study the effects of the cross-link on DNA structure or they can be ligated with larger DNA molecules to produce substrates for repair studies. This review will focus on examples of cross-linked duplexes that have been designed specifically to further our understanding of interstrand cross-link structure and repair.

Branchpoint sugar stereochemistry determines the hydrolytic susceptibility of branched RNA fragments by the yeast debranching enzyme (YDBR).

A series of branched RNAs (Y-shaped) related to yeast pre-mRNA splicing intermediates were synthesized incorporating both natural (i.e., ribose) and non-natural (i.e., arabinose, xylose and acyclic nucleoside) branchpoints in order to examine the effect of sugar conformation and phosphodiester configuration on yDBR hydrolytic efficiency. The results indicate that 2'-phosphodiester scission with yDBR occurs only with a cis-arrangement of phosphate groups at the branchpoint (i.e., ribose) thereby discriminating between all other configurations.

NMR structure of an ethylene interstrand cross-linked DNA which mimics the lesion formed by 1,3-bis(2-chloroethyl)-1-nitrosourea.

The bisalkylating agent 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), used in cancer chemotherapy to hinder cellular proliferation, forms lethal interstrand cross-links (ICLs) in DNA. BCNU generates an ethylene linkage connecting the two DNA strands at the N1 atom of 2'-deoxyguanosine and N3 atom of 2'-deoxycytidine, which is a synthetically challenging probe to prepare. To this end, an ICL duplex linking the N1 atom of 2'-deoxyinosine to the N3 atom of thymidine via an ethylene linker was devised as a mimic. We have solved the structure of this ICL duplex by a combination of molecular dynamics and high-field NMR experiments. The ethylene linker is well-accommodated in the duplex with minimal global and local perturbations relative to the unmodified duplex. These results may account for the substantial stabilization of the ICL duplex observed by UV thermal denaturation experiments and provides structural insights of a probe that may be useful for DNA repair studies.

Synthesis of building blocks and oligonucleotides containing {T}O4-alkylene-O4{T} interstrand cross-links.

This protocol describes the preparation of O(4)-thymidine-alkylene-O(4)-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 O(4)-thymidine-alkylene-O(4)-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.