In Vitro Ligation of Oligodeoxynucleotides Containing C8-Oxidized Purine Lesions Using Bacteriophage T4 DNA Ligase

Abstract

Ligases conduct the final stage of repair of DNA damage by sealing a single-stranded nick after excision of damaged nucleotides and reinsertion of correct nucleotides. Depending upon the circumstances and the success of the repair process, lesions may remain at the ligation site, either in the template or at the oligomer termini to be joined. Ligation experiments using bacteriophage T4 DNA ligase were carried out with purine lesions in four positions surrounding the nick site in a total of 96 different duplexes. The oxidized lesion 8-oxo-7,8-dihydroguanosine (OG) showed, as expected, that the enzyme is most sensitive to lesions on the 3′ end of the nick compared to the 5′ end and to lesions located in the intact template strand. In general, substrates containing the OG·A mismatch were more readily ligated than OG·C. Ligations of duplexes containing the OA·T base pair (OA=8-oxo-7,8-dihydroadenosine) that could adopt an anti-anti conformation proceeded in high efficiencies. An OI·Acontaining duplex (OI = 8-oxo-7,8-dihydroinosine) behaved similarly to OG·A. Due to its low reduction potential, OG is readily oxidized to secondary oxidation products, such as the guanidinohydantoin (Gh) and spiroiminodihydantoin (Sp) nucleosides; these lesions also contain an oxo group at the original C8 position of the purine. Ligation of oligomers containing Gh and Sp occurred when opposite A and G although the overall ligation efficiencies were much lower than most OG base pairs. Steady-state kinetic studies were carried out for representative examples of lesions in the template. K m increased by 90-100-fold for OG·C, OI·C, OI·A and OA·T containing duplexes compared to G·C. Substrates containing Gh·A, Gh·G, Sp·A and Sp·G base pairs showed K m values 20-70-fold higher than G·C while the K m value for OG·A was 5 times lower than G·C.Reactive oxygen species (ROS) 1 such as O 2 −•, HO• and H 2 O 2 are continuously produced during normal metabolic processes, and their production is augmented by inflammation and exposure to certain agents (1,2). DNA is sensitive to ROS, and in vivo oxidative damage results in DNA strand breaks, base modifications and DNA-protein cross-links (1). Unrepaired oxidative DNA damage can be mutagenic and is implicated in carcinogenesis, neurological disorders and aging (2-4). Oxidation of guanine, the most easily oxidized nucleobase (5), can lead to the commonly observed lesion 8-oxo-7,8-dihydro-2′-deoxyguanosine (OG) which is regarded as a biomarker of oxidative DNA damage in the cell (6). OG is mutagenic in the absence of repair leading to G→T transversions (7-10).OG can be removed by the base excision repair pathway (BER) (11-14) involving glycosidic bond cleavage of the damaged base followed by excision of the remaining abasic (AP) site (15). A nucleotide is inserted by a DNA polymerase, and in a final stage, the nick is sealed by a DNA ligase (16)(17)(18)(19)(20). Although DNA repair of oxidized base lesions has been extensively

Repair of Hydantoin Lesions and Their Amine Adducts in DNA by Base and Nucleotide Excision Repair

“…5,9 Appropriate oxidation conditions were used to produce oligonucleotides containing primarily Gh or Sp; subsequent separation of the desired lesion oligonucleotide from other products was performed using high performance liquid chromatography (HPLC). 52 A 33;nt fragment was used for the Ir(IV) oxidation reaction to make the Gh or Sp;containing oligonucleotide which was then ligated to a 17;nt strand using a 36;nt complement sequence as a template. 51 In this 33;nt sequence, the two diastereomers of Sp could not be separated via HPLC and therefore, the duplex substrates containing Sp are a mixture of diastereomers.…”

Reverse Transcription Past Products of Guanine Oxidation in RNA Leads to Insertion of A and C opposite 8-Oxo-7,8-dihydroguanine and A and G opposite 5-Guanidinohydantoin and Spiroiminodihydantoin Diastereomers

“…B - Structures of the base pairs between G or A and Gh or Sp are based on previous literature reports. 79, 80 …”

Products of Oxidative Guanine Damage Form Base Pairs with Guanine

“…The efficiency of incorporation of nucleotides opposite Gh is influenced by adjacent bases [ 36 ]. Moreover, the stabilities of Gh:G and Gh:A seem to depend on their positions in the sequence [ 37 ].…”

“…The T m values of Sp:A and Sp:G base pairs differ depending on the surrounding sequence [ 36 ]. Similarly, the result of experiments of T4 DNA ligation suggests that the stabilities of Sp:G and Sp:A depend on their position in the sequence [ 37 ].…”