Mechanism of action of a mammalian DNA repair endonuclease

Abstract

The mechanism of action of a DNA repair endonuclease isolated from calf thymus was determined. The calf thymus endonuclease possesses a substrate specificity nearly identical with that of Escherichia coli endonuclease III following DNA damage by high doses of UV light, osmium tetroxide, and other oxidizing agents. The calf thymus enzyme incises damaged DNA at sites of pyrimidines. A cytosine photoproduct was found to be the primary monobasic UV adduct. The calf thymus endonuclease and E. coli endonuclease III were found to possess similar, but not identical, DNA incision mechanisms. The mechanism of action of the calf thymus endonuclease was deduced by analysis of the 3' and 5' termini of the enzyme-generated DNA scission products with DNA sequencing methodologies and HPLC analysis of the material released by the enzyme following DNA damage. The calf thymus endonuclease removes UV light and osmium tetroxide damaged bases via an N-glycosylase activity followed by a 3' apurinic/apyrimidinic (AP) endonuclease activity. The calf thymus endonuclease also possesses a novel 5' AP endonuclease activity not possessed by endonuclease III. The product of this three-step mechanism is a nucleoside-free site flanked by 3'-and 5'-terminal phosphate groups. These results indicate the conservation of both substrate specificity and mechanism of action in the enzymatic removal of oxidative base damage between prokaryotes and eukaryotes. We propose the name redoxy endonucleases for this group of enzymes.

Specific Interaction of DNA Polymerase β and DNA Ligase I in a Multiprotein Base Excision Repair Complex from Bovine Testis

“…Uracil-DNA glycosylase (UDG), which catalyzes the removal of uracil from a G:U mismatch to initiate BER, is the most extensively studied of these enzymes (2)(3)(4). The apurinic/apyrimidinic (AP) site is generated by DNA glycosylase (5) and also by spontaneous cleavage of the glycosidic bond, in particular those involving purines (6). Both procaryotes and eucaryotes contain AP endonucleases (APE) that incise the phosphodiester backbone of DNA at the 5Ј side of an AP site leaving a 3Ј-OH and 5Ј-deoxyribose phosphate (5,7).…”

“…The apurinic/apyrimidinic (AP) site is generated by DNA glycosylase (5) and also by spontaneous cleavage of the glycosidic bond, in particular those involving purines (6). Both procaryotes and eucaryotes contain AP endonucleases (APE) that incise the phosphodiester backbone of DNA at the 5Ј side of an AP site leaving a 3Ј-OH and 5Ј-deoxyribose phosphate (5,7). Subsequently, the 5Ј-deoxyribose phosphate is removed either by a deoxyribose phosphodiesterase (dRpase) or by a 5Ј33Ј exonuclease, generating a onenucleotide gap with 3Ј-OH and 5Ј-phosphate termini (8 -11).…”

Dna Repair Enzymes

“…M. luteus mutants deficient in the pyrimidine dimer glycosylate activity apparently have normal UV resistance, because this organism has a nucleotide-excision-repair system as well (102 This enzyme was originally identified by its endonuclease activity on heavily UV-irradiated DNA . It has also been re ferred to as X-ray endonuclease, thymine glycol DNA glycosylase (6,8,100,103), and more recently, redoxyendonuclease (104). The enzyme has been purified to homogeneity (105).…”

Functional Analysis of the Amino-terminal 8-kDa Domain of DNA Polymerase β as Revealed by Site-directed Mutagenesis

“…Base excision repair was initially described in Escherichia coli (1) and later in mammalian cells (2). This repair pathway is initiated by enzymatic removal of an inappropriate base or spontaneous hydrolysis of bases through cleavage of the N-glycosyl bond (3,4). The resulting apurinic/apyrimidinic (AP) 1 site is cleaved by a class II AP endonuclease (5), which incises the phosphodiester backbone 5Ј to the AP site resulting in a 3Ј-hydroxyl and 5Ј 2-deoxyribose 5-phosphate (dRP) containing termini.…”