Multiply damaged sites in DNA: interactions with Escherichia coli endonucleases III and VIII

Abstract

Bursts of free radicals produced by ionization of water in close vicinity to DNA can produce clusters of opposed DNA lesions and these are termed multiply damaged sites (MDS). How MDS are processed by the Escherichia coli DNA glycosylases, endonuclease (endo) III and endo VIII, which recognize oxidized pyrimidines, is the subject of this study. Oligonucleotide substrates were constructed containing a site of pyrimidine damage or an abasic (AP) site in close proximity to a single nucleotide gap, which simulates a free radical-induced single-strand break. The gap was placed in the opposite strand 1, 3 or 6 nt 5' or 3' of the AP site or base lesion. Endos III and VIII were able to cleave an AP site in the MDS, no matter what the position of the opposed strand break, although cleavage at position one 5' or 3' was reduced compared with cleavage at positions three or six 5' or 3'. Neither endo III nor endo VIII was able to remove the base lesion when the gap was positioned 1 nt 5' or 3' in the opposite strand. Cleavage of the modified pyrimidine by endo III increased as the distance increased between the base lesion and the opposed strand break. With endo VIII, however, DNA breakage at the site of the base lesion was equivalent to or less when the gap was positioned 6 nt 3' of the lesion than when the gap was 3 nt 3' of the lesion. Gel mobility shift analysis of the binding of endo VIII to an oligonucleotide containing a reduced AP (rAP) site in close opposition to a single nucleotide gap correlated with cleavage of MDS substrates by endo VIII. If the strand break in the MDS was replaced by an oxidized purine, 7,8-dihydro-8-oxoguanine (8-oxoG), neither endo VIII cleavage nor binding were perturbed. These data show that processing of oxidized pyrimidines by endos III and VIII was strongly influenced by the position and type of lesion in the opposite strand, which could have a significant effect on the biological outcome of the MDS lesion.

Clustered DNA Damage, Influence on Damage Excision by XRS5 Nuclear Extracts and Escherichia coli Nth and Fpg Proteins

“…Therefore, it is suggested that a single-strand break on the non-labeled complementary strand at the AP site position is already formed prior to removal of the DHT. This finding is comparable with that for the removal of thymine glycol opposite a gap by Nth (10). However, the inhibitory effect of an AP site on the excision of thymine glycol is symmetrical in contrast to the asymmetry seen for excision of DHT opposite to an AP site by Nth.…”

“…There are only few studies about the excision of a damage substrate in the vicinity of another damage. In particular, synthesized oligonucleotides containing damage at specific sites were used to focus on the efficiencies of endonucleases VIII (Nei) and III (Nth) to excise either thymine glycol or DHT when opposite a singlestrand gap (10) as well as the efficiency of Fpg to excise 8-oxo-G or AP site opposite a gap (11) or 8-oxo-G near a formylamine on the same strand (12). Chaudhry and Weinfeld (13) focused on the efficiency of Nth to process either clustered abasic site or two DHTs on opposed strands within a plasmid.…”

Nucleosomes Suppress the Formation of Double-strand DNA Breaks during Attempted Base Excision Repair of Clustered Oxidative Damages

“…Construct names are in parentheses and indicate the lesion, top strand lesion position with respect to the dyad axis (Ϫ50 or Ϫ51), and base pair stagger (2 or 5) between opposing strands. The predicted yield of DSBs is based upon previous work using naked DNA substrates (38,62) as well as relative accessibility of the lesions with respect to the histone core. C, crystal structure of the 1.…”

Dynamics and diversions in base excision DNA repair of oxidized abasic lesions

“…However, the activity of DNA glycosylases such as the Fpg and Nth proteins is strongly sensitive to the presence of damage in the opposing strand (David-Cordonnier et al, 2001;Harrison et al, 1998). The human apurinic/ apyrimidinic (AP) endonuclease Ape1 is inhibited by a second abasic residue in the opposing strand 5' to its target AP site, but is unaffected by such lesions on the 3' side (Chaudhry et al, 1999;Strauss et al, 1997;Wilson et al, 1995).…”