The Action of DNA Ligase at Abasic Sites in DNA

Bogenhagen, Daniel F.; Pinz, Kevin G.

doi:10.1074/jbc.273.14.7888

Cited by 46 publications

(44 citation statements)

References 20 publications

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“…However, the overall fold of E23Q cv-pdg is probably correct because it did have some very minor activity on AP site-containing DNA; however, even this activity could be attributed to the presence of various basic residues on the face of this DNA binding enzyme in close proximity to the AP site. Similar observations have been made for DNA ligase on APcontaining DNAs (8).…”

Section: Figsupporting

confidence: 65%

The Catalytic Mechanism of a Pyrimidine Dimer-specific Glycosylase (pdg)/Abasic Lyase, chlorella virus-pdg

Garvish¹,

Lloyd²

1999

Journal of Biological Chemistry

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The repair of UV light-induced cyclobutane pyrimidine dimers can proceed via the base excision repair pathway, in which the initial step is catalyzed by DNA glycosylase/abasic (AP) lyases. The prototypical enzyme studied for this pathway is endonuclease V from the bacteriophage T4 (T4 bacteriophage pyrimidine dimer glycosylase (T4-pdg)). The first homologue for T4-pdg has been found in a strain of Chlorella virus (strain Paramecium bursaria Chlorella virus-1), which contains a gene that predicts an amino acid sequence homology of 41% with T4-pdg. Because both the structure and critical catalytic residues are known for T4-pdg, homology modeling of the Chlorella virus pyrimidine dimer glycosylase (cv-pdg) predicted that a conserved glutamic acid residue (Glu-23) would be important for catalysis at pyrimidine dimers and abasic sites. Sitedirected mutations were constructed at Glu-23 to assess the necessity of a negatively charged residue at that position (Gln-23) and the importance of the length of the negatively charged side chain (Asp-23). E23Q lost glycosylase activity completely but retained low levels of AP lyase activity. In contrast, E23D retained near wild type glycosylase and AP lyase activities on cis-syn dimers but completely lost its activity on the trans-syn II dimer, which is very efficiently cleaved by the wild type cv-pdg. As has been shown for other glyscosylases, the wild type cv-pdg catalyzes the cleavage at dimers or AP sites via formation of an imino intermediate, as evidenced by the ability of the enzyme to be covalently trapped on substrate DNA when the reactions are carried out in the presence of a strong reducing agent; in contrast, E23D was very poorly trapped on cis-syn dimers but was readily trapped on DNA containing AP sites. It is proposed that Glu-23 protonates the sugar ring, so that the imino intermediate can be formed.UV light damages DNA through the formation of two types of pyrimidine dimers: cyclobutane pyrimidine dimers and 6-4 photoproducts (1). One mechanism for the repair of the cyclobutane pyrimidine dimer is the base excision repair pathway, which is initiated by a DNA glycosylase/abasic (AP) 1 lyase.Although many DNA-containing viruses that have sustained UV-induced DNA damage, use host cell enzymes to repair their DNA, the bacteriophage T4 is unusual in that it encodes an enzyme, endonuclease V (T4-pdg, pyrimidine dimer glycosylase), that cleaves the N-glycosyl bond of the 5Ј thymine of the dimer and then subsequently cleaves the phosphodiester backbone, producing a ring opened sugar as an ␣, ␤ unsaturated aldehyde (2-4). T4-pdg has been characterized extensively since its discovery over 40 years ago, and its structure and mechanism of catalysis have been recently reviewed (5, 6).The first eukaryotic homologue of T4-pdg has been found to be encoded within the genome of an algal virus, Paramecium bursaria Chlorella virus-1. As a prelude to investigating structure-function relationships in Chlorella virus pyrimidine dimer glycosylase (cv-pdg), it is advantageous to utilize...

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Section: Figsupporting

confidence: 65%

The Catalytic Mechanism of a Pyrimidine Dimer-specific Glycosylase (pdg)/Abasic Lyase, chlorella virus-pdg

Garvish¹,

Lloyd²

1999

Journal of Biological Chemistry

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“…This represented the first observation of potential dRP lyase activity in a DNA polymerase other than DNA pol ␤, and the first observation of an AP lyase activity in a DNA ligase. We found that other ATP-dependent DNA ligases, including T4 and T7 DNA ligases, also contain AP lyase activity (18).…”

Section: Abasic (Ap)mentioning

confidence: 92%

Characterization of a Catalytically Slow AP Lyase Activity in DNA Polymerase γ and Other Family A DNA Polymerases

Pinz

Bogenhagen

2000

Journal of Biological Chemistry

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Mitochondrial DNA polymerase ␥ (pol ␥) is active in base excision repair of AP (apurinic/apyrimidinic) sites in DNA. Usually AP site repair involves cleavage on the 5 side of the deoxyribose phosphate by AP endonuclease. Previous experiments suggested that DNA pol ␥ acts to catalyze the removal of a 5-deoxyribose phosphate (dRP) group in addition to playing the conventional role of a DNA polymerase. We confirm that DNA pol ␥ is an active dRP lyase and show that other members of the family A of DNA polymerases including Escherichia coli DNA pol I also possess this activity. The dRP lyase reaction proceeds by formation of a covalent enzyme-DNA intermediate that is converted to an enzyme-dRP intermediate following elimination of the DNA. Both intermediates can be cross-linked with NaBH 4 . For both DNA pol ␥ and the Klenow fragment of pol I, the enzyme-dRP intermediate is extremely stable. This limits the overall catalytic rate of the dRP lyase, so that family A DNA polymerases, unlike pol ␤, may only be able to act as dRP lyases in repair of AP sites when they occur at low frequency in DNA. Abasic (AP)1 sites in DNA are produced frequently by spontaneous base loss or by the action of DNA glycosylases that initiate base excision repair of damaged bases in DNA (1, 2). If AP sites are not repaired quickly, DNA polymerases are capable of replicating through these non-instructional lesions, resulting in frequent misincorporation. Cells have adapted vigorous mechanisms for the repair of AP sites, usually beginning with incision of the phosphodiester backbone on the 5Ј side of the lesion by AP endonuclease to produce a 3Ј-OH terminus adjacent to a 5Ј-deoxyribose phosphate, or 5Ј-dRP group (3, 4). The 3Ј-OH terminus provides a primer for repair synthesis by DNA polymerase. The 5Ј-dRP moiety must be removed in the course of repair. This is frequently accomplished by a ␤-elimination reaction catalyzed by an AP lyase activity. When an AP lyase acts on an exposed 5Ј-dRP residue produced by a class II AP endonuclease, the activity may be considered as a dRP lyase. The dRP lyase mechanism involves nucleophilic attack on the C-1 position of the 5Ј-dRP group by a free amino group of the enzyme (5). This produces a transient covalent intermediate in which the DNA substrate is linked to the enzyme as a Schiff base that can be stabilized by treatment with strong reducing agents, such as sodium borohydride. This borohydride trapping reaction has been used to identify a number of DNA repair enzymes with AP lyase activity, including several DNA glycosylases (6, 7).Eukaryotic cells have redundant pathways for repairing AP sites in nuclear DNA. Under most circumstances, AP sites are repaired by a pathway that employs the dedicated repair polymerase, DNA pol ␤. Recently, Matsumoto and Kim (8) showed that the pol ␤ is especially well adapted to function in base excision repair, since it contains a dRP lyase activity in a small domain not required for polymerase activity. The active site of the pol ␤ dRP lyase has been localized to a heli...

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“…An antibody directed against the strictly mitochondrial cytochrome oxidase subunit IV (OX IV) was used to control for the enrichment of the mitochondrial fraction and to serve as a reference when comparing mitochondrial p53 levels in differently treated cultures. We have employed the same techniques (Bogenhagen and Clayton, 1974;Marchenko et al, 2000) in the following studies. Exponentially proliferating HCT116 cultures (10 8 cells) were mock treated or incubated with 1.36 mM ADR or 375 mM 5FU for 18 h. After cell fractionation, 5 mg each of total cell protein (t), mitochondrial (HM) fraction and free protein (f) fraction containing cytosolic plus leaked nuclear proteins were analysed by immunoblotting (Figure 3a).…”

Section: Both Adr and 5fu Induce P53's Presence In Mitochondrial Fracmentioning

confidence: 99%

“…Mitochondrial and free cytosolic protein fractions were prepared as reported before (Bogenhagen and Clayton, 1974;Marchenko et al, 2000). In brief, 10 8 cells were scraped into 5 ml ice-cold TD buffer (135 mM NaCl, 5 mM KCl, 25 mM TrisHCl pH 7.5), and 0.5 ml of the cell suspension was saved and dissolved in Laemmli buffer (50 mM Tris-HCl pH 6.8, 100 mM DTT, 2% SDS, 20% glycerol) as total cell protein.…”

Section: Preparation Of Subcellular Fractions and Immunoblot Analysismentioning

confidence: 99%

Mitochondrial p53 levels parallel total p53 levels independent of stress response in human colorectal carcinoma and glioblastoma cells

et al. 2004

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The Action of DNA Ligase at Abasic Sites in DNA

Cited by 46 publications

References 20 publications

The Catalytic Mechanism of a Pyrimidine Dimer-specific Glycosylase (pdg)/Abasic Lyase, chlorella virus-pdg

The Catalytic Mechanism of a Pyrimidine Dimer-specific Glycosylase (pdg)/Abasic Lyase, chlorella virus-pdg

Characterization of a Catalytically Slow AP Lyase Activity in DNA Polymerase γ and Other Family A DNA Polymerases

Mitochondrial p53 levels parallel total p53 levels independent of stress response in human colorectal carcinoma and glioblastoma cells

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