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Irreversible inhibition of DNA polymerase beta by an oxidized abasic lesion.

ABSTRACT: DNA damage is a source of carcinogenicity and is also the source of the cytotoxicity of gamma-radiolysis and antitumor agents, such as the enediynes. The dioxobutane lesion (DOB) is produced by a variety of DNA-damaging agents, including the aforementioned. Repair of DOB is important for maintaining the integrity of the genome as well as counteracting therapeutic agents that target DNA. We demonstrate that the DOB lesion efficiently and irreversibly inhibits repair by DNA polymerase beta (Pol beta), an integral enzyme in base-excision repair. Irreversible inhibition of Pol beta by DOB suggests that this lesion provides a chemical explanation for the cytotoxicity of drugs that produce it and explains previously unexplained observations in the literature concerning abasic lesions that are not repaired efficiently. Finally, these observations provide the impetus for the design of a new family of inhibitors of Pol beta.

SUBMITTER: Guan L

PROVIDER: S-EPMC2863033 | biostudies-literature | 2010 Apr

REPOSITORIES: biostudies-literature

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Irreversible inhibition of DNA polymerase beta by an oxidized abasic lesion.

Guan Lirui L Greenberg Marc M MM

Journal of the American Chemical Society 20100401 14

DNA damage is a source of carcinogenicity and is also the source of the cytotoxicity of gamma-radiolysis and antitumor agents, such as the enediynes. The dioxobutane lesion (DOB) is produced by a variety of DNA-damaging agents, including the aforementioned. Repair of DOB is important for maintaining the integrity of the genome as well as counteracting therapeutic agents that target DNA. We demonstrate that the DOB lesion efficiently and irreversibly inhibits repair by DNA polymerase beta (Pol be ...[more]

PMID: 20334373

Similar Datasets

DNA polymerase ? inactivation by oxidized abasic sites.

Project description:Base excision repair (BER) plays a vital role in maintaining genomic integrity in mammalian cells. DNA polymerase ? (Pol ?) is believed to play a backup role to DNA polymerase ? (Pol ?) in base excision repair. Two oxidized abasic lesions that are produced by a variety of DNA-damaging agents, including several antitumor antibiotics, the C4'-oxidized abasic site following Ape1 incision (pC4-AP), and 5'-(2-phosphoryl-1,4-dioxobutane) (DOB), irreversibly inactivate Pol ? and Pol ?. The interactions of DOB and pC4-AP with Pol ? are examined in detail using DNA substrates containing these lesions at defined sites. Single-turnover kinetic experiments show that Pol ? excises DOB almost 13 times more slowly than a 5'-phosphorylated 2-deoxyribose (dRP). pC4-AP is excised approximately twice as fast as DOB. The absolute rate constants are considerably slower than those reported for Pol ? for the respective reactions, suggesting that Pol ? may be an inefficient backup in BER. DOB inactivates Pol ? approximately 3-fold less efficiently than it does Pol ?, and the difference can be attributed to a higher K(I) (33 ± 7 nM). Inactivation of Pol ?'s lyase activity by DOB also prevents the enzyme from conducting polymerization following preincubation of the protein and DNA. Mass spectral analysis of GluC-digested Pol ? inactivated by DOB shows that Lys324 is modified. There is inferential support for the idea that Lys312 may also be modified. Both residues are within the Pol ? lyase active site. When acting on pC4-AP, Pol ? achieves approximately four turnovers on average before being inactivated. Lyase inactivation by pC4-AP is also accompanied by loss of polymerase activity, and mass spectrometry indicates that Lys312 and Lys324 are modified by the lesion. The ability of DOB and pC4-AP to inactivate Pol ? provides additional evidence that these lesions are significant sources of the cytotoxicity of DNA-damaging agents that produce them.

| S-EPMC3566640 | biostudies-literature

DNA polymerase V kinetics support the instructive nature of an oxidized abasic lesion in Escherichia coli.

Project description:Translesion synthesis past an oxidized abasic site, 2-deoxyribonolactone, in Escherichia coli results in high levels of dG incorporation and is dependent upon DNA polymerase V (Pol V). Kinetic experiments performed here affirm that Pol V preferentially incorporates dG opposite 2-deoxyribonolactone (L). Pol V discriminates between dG and dA on the basis of the apparent KD, suggesting that L provides instructive structural information to the enzyme despite lacking a Watson-Crick base.

| S-EPMC3817503 | biostudies-literature

Irreversible inhibition of DNA polymerase ? by small-molecule mimics of a DNA lesion.

Project description:Abasic sites are ubiquitous DNA lesions that are mutagenic and cytotoxic but are removed by the base excision repair pathway. DNA polymerase ? carries out two of the four steps during base excision repair, including a lyase reaction that removes the abasic site from DNA following incision of its 5'-phosphate. DNA polymerase ? is overexpressed in cancer cells and is a potential anticancer target. Recently, DNA oxidized abasic sites that are produced by potent antitumor agents were shown to inactivate DNA polymerase ?. A library of small molecules whose structures were inspired by the oxidized abasic sites was synthesized and screened for the ability to irreversibly inhibit DNA polymerase ?. One candidate (3a) was examined more thoroughly, and modification of its phosphate backbone led to a molecule that irreversibly inactivates DNA polymerase ? in solution (IC50 ? 21 ?M), and inhibits the enzyme's lyase activity in cell lysates. A bisacetate analogue is converted in cell lysates to 3a. The bisacetate is more effective in cell lysates, more cytotoxic in prostate cancer cells than 3a and potentiates the cytotoxicity of methyl methanesulfonate between 2- and 5-fold. This is the first example of an irreversible inhibitor of the lyase activity of DNA polymerase ? that works synergistically with a DNA damaging agent.

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Excision of a lyase-resistant oxidized abasic lesion from DNA.

Project description:The C2'-oxidized abasic lesion (C2-AP) is produced in DNA that is subjected to oxidative stress. The lesion disrupts replication and gives rise to mutations that are dependent upon the identity of the upstream nucleotide. Ape1 incises C2-AP, but the 5'-phosphorylated fragment is not a substrate for the lyase activity of DNA polymerase beta. Excision of the lesion is achieved by strand displacement synthesis in the presence of flap endonuclease during which C2-AP and the 3'-adjacent nucleotide are replaced. The oxidized abasic lesion is also a substrate for the bacterial UvrABC nucleotide excision repair system. These data suggest that the redundant nature of DNA repair systems provides a means for removing a lesion that resists excision by short patch base excision repair.

| S-EPMC2863051 | biostudies-literature

DNA synthesis across an abasic lesion by yeast REV1 DNA polymerase.

Project description:Abasic (apurinic/apyrimidinic) sites are among the most abundant DNA lesions in humans, and they present a strong block to replication. They are also highly mutagenic because when replicative DNA polymerases manage to insert a nucleotide opposite the lesion, they prefer to insert an A. Rev1, a member of Y-family DNA polymerases, does not obey the A-rule. This enzyme inserts a C opposite an abasic lesion with much greater catalytic efficiency than an A, G, or T. We present here the structure of yeast Rev1 in ternary complex with DNA containing an abasic lesion and with dCTP as the incoming nucleotide. The structure reveals a mechanism of synthesis across an abasic lesion that differs from that in other polymerases. The lesion is driven to an extrahelical position, and the incorporation of a C is mediated by an arginine (Arg324) that is conserved in all known orthologs of Rev1, including humans. The hydrophobic cavity that normally accommodates the unmodified G is instead filled with water molecules. Since Gs are especially prone to depurination through a spontaneous hydrolysis of the glycosidic bond, the ability of Rev1 to stabilize an abasic lesion in its active site and employ a surrogate arginine to incorporate a C provides a unique means for the "error-free" bypass of this noninstructional lesion.

| S-EPMC3127253 | biostudies-literature

The A-Rule and Deletion Formation During Abasic and Oxidized Abasic Site Bypass by DNA Polymerase ?.

Project description:DNA polymerase ? (Pol ?) is implicated in various cellular processes including double-strand break repair and apurinic/apyrimidinic site bypass. Because Pol ? expression correlates with poor cancer prognosis, the ability of Pol ? to bypass the C4'-oxidized abasic site (C4-AP) and 2-deoxyribonolactone (L), which are generated by cytotoxic agents, is of interest. Translesion synthesis and subsequent extension by Pol ? past C4-AP or L and an abasic site (AP) or its tetrahydrofuran analogue (F) was examined. Pol ? conducts translesion synthesis on templates containing AP and F with similar efficiencies and follows the "A-rule," inserting nucleotides in the order A > G > T. Translesion synthesis on templates containing C4-AP and L is less efficient than AP and F, and the preference for A insertion is reduced for L and absent for C4-AP. Extension past all abasic lesions (AP, F, C4-AP, and L) was significantly less efficient than translesion synthesis and yielded deletions caused by the base one or two nucleotides downstream from the lesion being used as a template, with the latter being favored. These results suggest that bypass of abasic lesions by Pol ? is highly mutagenic.

| S-EPMC5499511 | biostudies-literature

Human DNA polymerase beta mutations allowing efficient abasic site bypass.

Project description:The DNA of every cell in the human body gets damaged more than 50,000 times a day. The most frequent damages are abasic sites. This kind of damage blocks proceeding DNA synthesis by several DNA polymerases that are involved in DNA replication and repair. The mechanistic basis for the incapability of these DNA polymerases to bypass abasic sites is not clarified. To gain insights into the mechanistic basis, we intended to identify amino acid residues that govern for the pausing of DNA polymerase ? when incorporating a nucleotide opposite to abasic sites. Human DNA polymerase ? was chosen because it is a well characterized DNA polymerase and serves as model enzyme for studies of DNA polymerase mechanisms. Moreover, it acts as the main gap-filling enzyme in base excision repair, and human tumor studies suggest a link between DNA polymerase ? and cancer. In this study we employed high throughput screening of a library of more than 11,000 human DNA polymerase ? variants. We identified two mutants that have increased ability to incorporate a nucleotide opposite to an abasic site. We found that the substitutions E232K and T233I promote incorporation opposite the lesion. In addition to this feature, the variants have an increased activity and a lower fidelity when processing nondamaged DNA. The mutations described in this work are located in well characterized regions but have not been reported before. A crystallographic structure of one of the mutants was obtained, providing structural insights.

| S-EPMC3030401 | biostudies-literature

Mutagenic Bypass of an Oxidized Abasic Lesion-Induced DNA Interstrand Cross-Link Analogue by Human Translesion Synthesis DNA Polymerases.

Project description:5'-(2-Phosphoryl-1,4-dioxobutane) (DOB) is an oxidized abasic site that is produced by several antitumor agents and ?-radiolysis. DOB reacts reversibly with a dA opposite the 3'-adjacent nucleotide to form DNA interstrand cross-links (ICLs), genotoxic DNA lesions that can block DNA replication and transcription. Translesion synthesis (TLS) is an important step in several ICL repair pathways to bypass unhooked intermediates generated by endonucleolytic incision. The instability of DOB-ICLs has made it difficult to learn about their TLS-mediated repair capability and mutagenic potential. We recently developed a method for chemically synthesizing oligonucleotides containing a modified DOB-ICL analogue. Herein, we examined the capabilities of several highly relevant eukaryotic TLS DNA polymerases (pols), including human pol ?, pol ?, pol ?, pol ?, REV1, and yeast pol ?, to bypass this DOB-ICL analogue. The prelesion, translesion, and postlesion replication efficiency and fidelity were examined. Pol ? showed moderate bypass activity when encountering the DOB-ICL, giving major products one or two nucleotides beyond the cross-linked template nucleotide. In contrast, DNA synthesis by the other pols was stalled at the position before the cross-linked nucleotide. Steady-state kinetic data and liquid chromatography-mass spectrometry sequencing of primer extension products by pol ? unambiguously revealed that pol ?-mediated bypass is highly error-prone. Together, our study provides the first set of in vitro evidence that the DOB-ICL is a replication-blocking and highly miscoding lesion. Compared to several other TLS pols examined, pol ? is likely to contribute to the TLS-mediated repair of the DOB-ICL in vivo.

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Structural basis of transcriptional stalling and bypass of abasic DNA lesion by RNA polymerase II.

Project description:Abasic sites are among the most abundant DNA lesions and interfere with DNA replication and transcription, but the mechanism of their action on transcription remains unknown. Here we applied a combined structural and biochemical approach for a comprehensive investigation of how RNA polymerase II (Pol II) processes an abasic site, leading to slow bypass of lesion. Encounter of Pol II with an abasic site involves two consecutive slow steps: insertion of adenine opposite a noninstructive abasic site (the A-rule), followed by extension of the 3'-rAMP with the next cognate nucleotide. Further studies provided structural insights into the A-rule: ATP is slowly incorporated into RNA in the absence of template guidance. Our structure revealed that ATP is bound to the Pol II active site, whereas the abasic site is located at an intermediate state above the Bridge Helix, a conserved structural motif that is cirtical for Pol II activity. The next extension step occurs in a template-dependent manner where a cognate substrate is incorporated, despite at a much slower rate compared with nondamaged template. During the extension step, neither the cognate substrate nor the template base is located at the canonical position, providing a structural explanation as to why this step is as slow as the insertion step. Taken together, our studies provide a comprehensive understanding of Pol II stalling and bypass of the abasic site in the DNA template.

| S-EPMC5856558 | biostudies-literature

Long patch base excision repair compensates for DNA polymerase ? inactivation by the C4'-oxidized abasic site.

Project description:The C4'-oxidized abasic site (C4-AP), which is produced by a variety of damaging agents, has significant consequences for DNA. The lesion is highly mutagenic and reactive, resulting in interstrand cross-links. The base excision repair of DNA containing independently generated C4-AP was examined. C4-AP is incised by Ape1 ~12-fold less efficiently than an apurinic/apyrimidinic lesion. DNA polymerase ? induces the ?-elimination of incised C4-AP in ternary complexes, duplexes, and single-stranded substrate. However, excision from a ternary complex is most rapid. In addition, the lesion inactivates the enzyme after approximately seven turnovers on average by reacting with one or more lysine residues in the lyase active site. Unlike 5'-(2-phosphoryl-1,4-dioxobutane), which very efficiently irreversibly inhibits DNA polymerase ?, the lesion is readily removed by strand displacement synthesis conducted by the polymerase in conjunction with flap endonuclease 1. DNA repair inhibition by C4-AP may be a partial cause of the cytotoxicity of drugs that produce this lesion.

| S-EPMC3136615 | biostudies-literature

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