Derek K. O'Flaherty

Steady‐State Kinetic Analysis of DNA Polymerase Single‐Nucleotide Incorporation Products

This unit describes the experimental procedures for the steady‐state kinetic analysis of DNA synthesis across DNA nucleotides (native or modified) by DNA polymerases. In vitro primer extension experiments with a single nucleoside triphosphate species followed by denaturing polyacrylamide gel electrophoresis of the extended products is described. Data analysis procedures and fitting to steady‐state kinetic models is presented to highlight the kinetic differences involved in the bypass of damaged versus undamaged DNA. Moreover, explanations concerning problems encountered in these experiments are addressed. This approach provides useful quantitative parameters for the processing of damaged DNA by DNA polymerases.

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.

UNIT 5.17 Preparation of Intrastrand {G}O6-Alkylene-O6{G} Cross-Linked Oligonucleotides

This unit describes the preparation O 6‐2′‐deoxyguanosine‐butylene‐O 6‐2′‐deoxyguanosine dimer phosphoramidites and precursors for incorporation of site‐specific intrastrand cross‐links (IaCL) into DNA oligonucleotides. Protected 2′‐deoxyguanosine dimers are produced using the Mitsunobu reaction. IaCL DNA containing the intradimer phosphodiester are first chemically phosphorylated, followed by a ring‐closing reaction using the condensing reagent 1‐(2‐mesitylenesulfonyl)‐3‐nitro‐1H‐1,2,4‐triazole. Phosphoramidites are incorporated into oligonucleotides by solid‐phase synthesis and standard deprotection and cleavage protocols are employed. This approach allows for the preparation of IaCL DNA substrates in amounts and purity amenable for biophysical characterization, and biochemical studies as substrates to investigate DNA repair and bypass pathways.

Preparation of Intrastrand {G}O6‐Alkylene‐O6{G} Cross‐Linked Oligonucleotides

This unit describes the preparation O 6‐2′‐deoxyguanosine‐butylene‐O 6‐2′‐deoxyguanosine dimer phosphoramidites and precursors for incorporation of site‐specific intrastrand cross‐links (IaCL) into DNA oligonucleotides. Protected 2′‐deoxyguanosine dimers are produced using the Mitsunobu reaction. IaCL DNA containing the intradimer phosphodiester are first chemically phosphorylated, followed by a ring‐closing reaction using the condensing reagent 1‐(2‐mesitylenesulfonyl)‐3‐nitro‐1H‐1,2,4‐triazole. Phosphoramidites are incorporated into oligonucleotides by solid‐phase synthesis and standard deprotection and cleavage protocols are employed. This approach allows for the preparation of IaCL DNA substrates in amounts and purity amenable for biophysical characterization, and biochemical studies as substrates to investigate DNA repair and bypass pathways.

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. Curr. Protoc. Nucleic Acid Chem. 55:5.13.1‐5.13.19.