In vitro replication and repair of DNA containing a C2'-oxidized abasic site.
Abasic lesions are unable to form Watson-Crick hydrogen bonds with nucleotides. Nonetheless, polymerase and repair enzymes distinguish between various oxidized abasic lesions, as well as from nonoxidized abasic sites (AP). The C2-AP lesion is produced when DNA is exposed to gamma-radiolysis. Its effects on polymerases and repair enzymes are unknown. A recently reported method for the chemical synthesis of oligonucleotides containing C2-AP at a defined site was utilized for studying the activity of Klenow exo(-) and repair enzymes on templates containing the lesion. The C2-AP lesion has a similar effect on Klenow exo(-) as do AP and C4-AP sites. Deoxyadenosine is preferentially incorporated opposite C2-AP, but extension of the primer past the lesion is strongly blocked. C2-AP is incised less efficiently by exonuclease III and endonuclease IV than are other abasic lesions. Furthermore, although a Schiff base between C2-AP and endonuclease III can be chemically trapped, the location of the 3'-phosphate alpha with respect to the aldehyde prevents beta-elimination associated with the lyase activity of type I base excision repair enzymes. The interactions of the C2'-oxidized abasic site with Klenow exo(-) and repair enzymes suggest that the lesion will be mutagenic and that it will be removed by strand displacement synthesis and flap endonuclease processing via a long patch repair mechanism.
Greenberg
Marc M
MM
Department of Chemistry, The Johns Hopkins University, 3400 North Charles Street, Baltimore, Maryland 21218, USA. mgreenberg@jhu.edu
Weledji
Yvonne N
YN
Kroeger
Kelly M
KM
Kim
Jaeseung
J
D001080:Apurinic Acid / Q000138:chemical synthesis / Q000235:genetics / Q000378:metabolism*; D004256:DNA Polymerase I / Q000737:chemistry / Q000378:metabolism; D017931:DNA Primers / Q000138:chemical synthesis / Q000378:metabolism; D004260:DNA Repair; D004261:DNA Replication; D004269:DNA, Bacterial / Q000138:chemical synthesis / Q000235:genetics / Q000378:metabolism*; D043223:Deoxyribonuclease IV (Phage T4-Induced) / Q000737:chemistry / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism; D005090:Exodeoxyribonucleases / Q000737:chemistry / Q000378:metabolism; D009692:Nucleic Acid Heteroduplexes / Q000138:chemical synthesis / Q000235:genetics / Q000378:metabolism; D009693:Nucleic Acid Hybridization; D009841:Oligonucleotides / Q000138:chemical synthesis / Q000235:genetics / Q000378:metabolism; D010084:Oxidation-Reduction; D011119:Polynucleotides / Q000138:chemical synthesis / Q000235:genetics / Q000378:metabolism*; D012545:Schiff Bases / Q000737:chemistry / Q000378:metabolism; D013698:Templates, Genetic
D017931:DNA Primers; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D009692:Nucleic Acid Heteroduplexes; D009841:Oligonucleotides; D011119:Polynucleotides; D012545:Schiff Bases; C001790:apyrimidinic acid; D001080:Apurinic Acid; D004256:DNA Polymerase I; D005090:Exodeoxyribonucleases; C020133:exodeoxyribonuclease III; D043223:Deoxyribonuclease IV (Phage T4-Induced); C468972:endonuclease IV, E coli