Synthesis and Thermodynamic Studies of Oligonucleotides Containing the Two Isomers of Thymine Glycol

Clustered DNA damage, where two or more lesions are located proximally to each other, is frequently induced by ionizing radiation. Individual base lesions within a cluster are repaired by base excision repair. In this study we addressed the question of how thymine glycol (Tg) within a cluster would affect the repair of opposing lesions by human cell extracts. We have found that Tg located opposite to an abasic site does not affect cleavage of this site by apurinic/apyrimidinic (AP) endonuclease. However, Tg significantly compromised the next step of the repair. Although purified DNA polymerase ␤ was able to incorporate the correct nucleotide (dAMP) opposite to Tg, the rate of incorporation was reduced by 3-fold. Tg does not affect 5-sugar phosphate removal by the 2-deoxyribose-5-phosphate (dRP) lyase activity of DNA polymerase ␤, but further processing of the strand break by purified DNA ligase III was slightly diminished. In agreement with these findings, although an AP site located opposite to Tg was efficiently incised in human cell extract, only a limited amount of fully repaired product was observed, suggesting that such clustered DNA lesions may have a significantly increased lifetime in human cells compared with similar singlestanding lesions.Numerous cytotoxic agents exert their deleterious effects via the formation of lesions and adducts in DNA; these effects may include oxidized purine and pyrimidine residues, abasic sites, and single and double strand breaks. Ionizing radiation induces damage in DNA by direct ionization and through the generation of hydroxyl radicals that attack DNA, resulting in single strand breaks and oxidative damage to sugar and base residues (1). Two or more DNA lesions of the same or different nature may be produced proximal to each other on the same or opposite DNA strands, generally within two helical turns of the DNA. These various types of DNA damage, known as "clustered DNA lesions," may include strand breaks containing damaged DNA termini accompanied by multiple base lesions of varying complexity (2, 3).Approximately 10 -20% of the damage to DNA induced by ionizing radiation is the result of thymine base oxidation and fragmentation (4). Thymine is an easily oxidized base and is frequently found as a component of clustered lesions (2, 3). Individual base damages within a cluster are repaired by base excision repair (BER).1 BER is a multiprotein pathway with a broad substrate specificity that is determined by the damagespecific glycosylases. DNA glycosylases initiate BER by recognizing damaged or abnormal bases and cleaving the glycosylic bond linking the base to the sugar phosphate backbone (5). The majority of the apurinic/apyrimidinic sites (AP sites) formed are further processed by the so-called "short-patch" BER pathway (6). In human cells this pathway is activated by an AP endonuclease (APE1) that introduces a DNA strand break 5Ј to the AP site (7). This strand break cannot be ligated directly; therefore, DNA polymerase ␤ (Pol ␤) first adds one nucleotide to the 3Ј-en...

Section: Fig 3 Effect Of Thymine Glycol On Removal Of the Drp Residmentioning

confidence: 48%

Mode of Inhibition of Short-patch Base Excision Repair by Thymine Glycol within Clustered DNA Lesions

Budworth¹,

Dianov²

2003

“…50 and 51) particularly when canonical hydrogen bonding and/or stacking interactions are absent. Such a mechanism has been implied for AP sites and thymine glycol on the basis of the lack of apparent correlations between the preference of insertion or proofreading excision of nucleotides and the T m of oligonucleotides containing these lesions (52,53). The mechanism involving steric exclusion also seems consistent with the observation that Endo III recognizes mFapyG paired with purines more efficiently than that paired with pyrimidines (activity with respect to the paired base: A Ϸ G Ͼ T Ͼ C) (22).…”

Section: Figmentioning

confidence: 64%

Effects of a Guanine-derived Formamidopyrimidine Lesion on DNA Replication

Asagoshi

Terato

Ohyama

et al. 2002

2,6-Diamino-4-hydroxy-5-formamidopyrimidine derived from guanine (FapyG) is a major DNA lesion formed by reactive oxygen species. In this study, a defined oligonucleotide template containing a 5-N-methylated analog of FapyG (mFapyG) was prepared, and its effect on DNA replication was quantitatively assessed in vitro. The results were further compared with those obtained for 7,8-dihydro-8-oxoguanine and an apurinic/ apyrimidinic site embedded in the same sequence context. mFapyG constituted a fairly strong but not absolute block to DNA synthesis catalyzed by Escherichia coli DNA polymerase I Klenow fragment with and without an associated 3-5 exonuclease activity, thereby permitting translesion synthesis with a limited efficiency. The efficiency of translesion synthesis was G > 7,8-dihydro-8-oxoguanine > mFapyG > apurinic/apyrimidinic site. Analysis of the nucleotide insertion (f ins ‫؍‬ V max /K m for insertion) and extension (f ext ‫؍‬ V max /K m for extension) efficiencies for mFapyG revealed that the extension step constituted a major kinetic barrier to DNA synthesis. When mFapyG was bypassed, dCMP, a cognate nucleotide, was preferentially inserted opposite the lesion (dCMP (relative f ins ‫؍‬ 1) > > dTMP (2.4 ؋ 10 ؊4 ) Ϸ dAMP (8.1 ؋ 10 ؊5 ) > dGMP (4.5 ؋ 10 ؊7 )), and the primer terminus containing a mFapyG:C pair was most efficiently extended (mFapyG:C (relative f ext ‫؍‬ 1) > mFapyG:T (4.6 ؋ 10 ؊3 ) > > mFapyG:A and mFapyG:G (extension not observed)). Thus, mFapyG is a potentially lethal but not premutagenic lesion.Reactive oxygen species formed by aerobic metabolism, phagocytic blood cells upon inflammation, ionizing radiation, and photosensitized reactions generate structurally diverse oxidative damage to DNA that stores vital genetic information of cells (1-3). Base lesions thus formed are generally restored by the base excision repair pathway involving multiple enzymes such as N-glycosylase/AP 1 lyase, AP endonuclease, DNA polymerase, and DNA ligase (4, 5). However, if left unrepaired, they result in mutations and/or cell death. It has also been implied that oxidative DNA damage is involved in carcinogenesis and various degenerative diseases (6, 7). 7,8-Dihydro-8-oxoguanine (8-oxoG) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine derived from guanine (FapyG) have been identified as major products in the reaction of DNA with reactive oxygen species (Fig. 1A) (1, 2). When formed in DNA, 8-oxoG in a template directs incorporation of non-cognate dAMP as well as cognate dCMP during translesion synthesis by DNA polymerases, thereby inducing GC-to-TA transversions (8, 9). Similarly, when formed in a cellular nucleotide pool, a 2Ј-deoxyribonucleotide form of 8-oxoG can be incorporated opposite adenine as well as cytosine in a DNA template, inducing AT-to-CG transversions (10 -12). Mispair formation between 8-oxoG and adenine leading to mutation involves an unusual syn-conformer of 8-oxoG that can form two hydrogen bonds between O-6 and N-7-H in 8-oxoG and N-6 -H and N-1 in adenine without introducing significan...

“…Tg lesions are a strong block to many DNA polymerases in vitro and in vivo (34). Neither isomer of thymine glycol can form a stable base pair with any nucleobase on the complementary strand (35), and DNA synthesis terminates after insertion of A opposite Tg. For example, in our experiments, DNA synthesis by the B-family DNA polymerase from RB69 (gp43) was blocked by either Tg isomer.…”

Section: Discussionmentioning

confidence: 99%

Human DNA Polymerase N (POLN) Is a Low Fidelity Enzyme Capable of Error-free Bypass of 5S-Thymine Glycol

Takata

Shimizu

Iwai

et al. 2006

Self Cite

139

206

Human DNA polymerase N (POLN or pol ) is the most recently discovered nuclear DNA polymerase in the human genome. It is an A-family DNA polymerase related to Escherichia coli pol I, human POLQ, and Drosophila Mus308. We report the first purification of the recombinant enzyme and examination of its biochemical properties, as a step toward understanding the functions of POLN. Unusual for an A-family DNA polymerase, POLN is a low fidelity enzyme incorporating T opposite template G with a frequency of 0.45 and G opposite template T with a frequency of 0.021. The frequency of misincorporation of T opposite template G is higher than any other known DNA polymerase. POLN has a processivity of DNA synthesis (1-100 nucleotides) similar to the exonuclease-deficient Klenow fragment of E. coli pol I, is inhibited by dideoxynucleotides, and resistant to aphidicolin. The strand displacement activity of POLN was higher than exonuclease-deficient Klenow fragment. Furthermore, POLN can perform translesion synthesis past thymine glycol, a common endogenous and radiationinduced product of reactive oxygen species damage to DNA. Thymine glycol blocks DNA synthesis by most DNA polymerases, but POLN was particularly adept at efficient and accurate translesion synthesis past a 5S-thymine glycol.The human genome contains 15 distinct known DNA polymerase genes, and these are classified into four families A, B, X, and Y based on their amino acid sequences (1). Human DNA polymerase N (POLN), 2 the most recently discovered nuclear DNA polymerase, is an A-family enzyme with unknown function. The gene on chromosome 4p16.2 encodes a protein of 900 amino acid residues with a molecular mass of 100 kDa (2). The prototypical A-family DNA polymerase, Escherichia coli pol I is a high fidelity DNA polymerase that contributes to the maturation of Okazaki fragments during DNA replication and in gap-filling during base excision repair (BER), nucleotide excision repair (NER), and repair of DNA interstrand cross-links.Human POLQ, another A-family DNA polymerase, is similar to the Drosophila nuclear DNA polymerase Mus308 (3) in that it encodes both a DNA/RNA helicase domain and an A-family DNA polymerase domain (4, 5). By contrast, POLN has only the DNA polymerase domain. The POLN gene is encoded only in vertebrate genomes, but not in invertebrates or any lower eukaryotes. Possibly POLN has a role related to organ systems that are especially developed in vertebrates, such as the adaptive immune system or the brain. Expression studies of POLN are limited, but expression of the gene has been detected by Northern blotting in testes, heart and skeletal muscle tissue (2) and by expression sequence tagging in prostate, muscle, brain, and other organs. In cells from a human neuroblastoma patient, a chromosome fusion (1, 4) disrupting the DNA polymerase domain coding sequence of POLN was observed at diagnosis and at relapse. A (4, 17) fusion was detected at relapse only (6). It is possible that POLN might serve as a tumor suppressor in some cell types and that loss...