DNA polymerase beta null mouse embryonic fibroblasts harbor a homozygous null mutation in DNA polymerase iota

Sobol, Robert W.

doi:10.1016/j.dnarep.2006.08.005

Cited by 22 publications

(28 citation statements)

References 60 publications

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“…1 and data not shown). This is essentially in agreement with previously published data from another laboratory [18]. Since MEF cells deficient in BER are hypersensitive to alkylating agent-induced DNA damage, we next determined the resistance of MEFs to the monofunctional alkylating agent MMS.…”

supporting

confidence: 79%

Negligible impact of pol ι expression on the alkylation sensitivity of pol β-deficient mouse fibroblast cells

et al. 2008

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Dear EditorDNA repair plays a major role in maintaining genomic integrity such that deficiency, as well as an excess of DNA repair, has a significant impact. For example, the absence of DNA polymerase β(pol β) expression in mice results in embryonic lethality, while over-expression of pol β causes cataracts [1,2]. Alkylated DNA bases in higher eukaryotes are repaired predominantly by the base excision repair (BER) pathway. During "single-nucleotide" BER, an alkylated base is removed by the damage specific glycosylase, 3-methyladenine DNA glycosylase, and the resulting abasic site is cleaved by apurinic/apyrimidinic endonuclease. This results in a nick with a 5' dRP group that can be removed by the dRP lyase activity of pol β; the same polymerase then fills the single-nucleotide gap. Finally, a DNA ligase seals the nick completing repair (reviewed in [3]).Mouse embryonic fibroblasts (MEFs) deficient in pol β demonstrate increased sensitivity to alkylating agents such as methyl methanesulfonate (MMS) [4]. Hypersensitivity is moderate, and cellular extracts from pol β null cells have some residual BER activity, suggesting that other polymerases could participate in BER. Reversal of the MMS hypersensitivity requires the dRP lyase activity of pol β [5]. DNA polymerase λ(pol λ) also has dRP lyase activity and can function as a back up BER enzyme in pol β null cells [6,7].Another DNA polymerase carrying dRP lyase activity is DNA polymerase ι(pol ι) [7,8]. This polymerase is a member of the Y-family of DNA polymerases and was identified due to its homology with DNA polymerase η(pol η) [9]. Based on in vitro biochemical data and on the structure of the enzyme, it has been proposed that pol ι participates in DNA translesion synthesis during bypass of a damaged template [10,11]. Gene expression at the mRNA level is modest in most tissues, with strongest expression in testis and ovarian cells [9]. The data on the biological roles of this enzyme are contradictory. Pol ι is expressed at a high level in some human cancer cells and also is associated with increased frequency of mutagenesis in certain cell types; lowering the expression of this enzyme decreased mutation frequency [12]. Deletion of the pol ι gene in human Burkitt's lymphoma cells reduced inducible somatic hypermutation (SHM), and this process could be restored by expression of pol ι [13,14]. At the same time, a *Corresponding author. Tel.: 919-541-3267; Fax: 919-541-3592, E-mail address: wilson5@niehs.nih.gov (S.H. Wilson). Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public AccessAuthor Manuscript DNA Repair (Amst...

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confidence: 79%

Negligible impact of pol ι expression on the alkylation sensitivity of pol β-deficient mouse fibroblast cells

et al. 2008

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“…This mechanism or process of complex formation appears to provide an increase in specificity and efficiency to the BER pathway, thereby facilitating the maintenance of genome integrity by preventing the accumulation of highly toxic repair intermediates [83]. Decreased concentrations of just one protein within this pathway could significantly alter the balance of complex formation, resulting in reduced repair capacity and an increased exposure to toxic BER intermediates, such as has been observed in pol ß deficiency in mouse cells [79,[91][92][93], in animal models [94] and in human cells [62].…”

Section: Ber Protein Post-translational Modificationsmentioning

confidence: 97%

A unified view of base excision repair: Lesion-dependent protein complexes regulated by post-translational modification

Almeida¹,

Sobol²

2007

DNA Repair

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374

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Base excision repair (BER) proteins act upon a significantly broad spectrum of DNA lesions that result from endogenous and exogenous sources. Multiple sub-pathways of BER (short-path or longpatch) and newly designated DNA repair pathways (e.g., SSBR and NIR) that utilize BER proteins complicate any comprehensive understanding of BER and its role in genome maintenance, chemotherapeutic response, neurodegeneration, cancer or aging. Herein, we propose a unified model of BER, comprised of three functional processes: Lesion Recognition/Strand Scission, Gap Tailoring and DNA Synthesis/Ligation, each represented by one or more multiprotein complexes and coordinated via the XRCC1/DNA Ligase III and PARP1 scaffold proteins. BER therefore may be represented by a series of repair complexes that assemble at the site of the DNA lesion and mediates repair in a coordinated fashion involving protein-protein interactions that dictate subsequent steps or sub-pathway choice. Complex formation is influenced by post-translational protein modifications that arise from the cellular state or the DNA damage response, providing an increase in specificity and efficiency to the BER pathway. In this review, we have summarized the reported BER proteinprotein interactions and protein post-translational modifications and discuss the impact on DNA repair capacity and complex formation.

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“…DNA Pol ␤ has long been considered a DNA repair polymerase (Sobol, 2007). Pol ␤ is a 335-amino acid, bifunctional enzyme, facilitating both the DNA synthesis step in BER as figure) in 96-well plates for 24 h before exposure to 3-MA.…”

Section: Discussionmentioning

confidence: 99%

Human Methyl Purine DNA Glycosylase and DNA Polymerase β Expression Collectively Predict Sensitivity to Temozolomide

Trivedi

Wang²,

Jelezcova³

et al. 2008

Mol Pharmacol

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103

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Overexpression of N-methylpurine DNA glycosylase (MPG) has been suggested as a possible gene therapy approach to sensitize tumor cells to the cell-killing effects of temozolomide, an imidazotetrazine-class chemotherapeutic alkylating agent. In the present study, we show that both elevated MPG expression and short hairpin RNA-mediated loss of DNA polymerase ␤ (Pol ␤) expression in human breast cancer cells increases cellular sensitivity to temozolomide. Resistance to temozolomide is restored by complementation of either wild-type human Pol ␤ or human Pol ␤ with an inactivating mutation specific to the polymerase active site yet functional for 5Ј-deoxyribosephosphate (5ЈdRP) lyase activity. These genetic and cellular studies uniquely demonstrate that overexpression of MPG causes an imbalance in base excision repair (BER), leading to an accumulation of cytotoxic 5ЈdRP lesions, and that the 5ЈdRP lyase activity of Pol ␤ is required to restore resistance to temozolomide. These results imply that Pol ␤-dependent 5ЈdRP lyase activity is the rate-limiting step in BER in these cells and suggests that BER is a tightly balanced pathway for the repair of alkylated bases such as N7-methylguanine and N3-methyladenine. Furthermore, we find that 5ЈdRP-mediated cell death is independent of caspase-3 activation and does not induce the formation of autophagosomes, as measured by green fluorescent protein-light chain 3 localization. The experiments presented herein suggest that it will be important to investigate whether an active BER pathway could be partially responsible for the temozolomide-mediated resistance seen in some tumors and that balanced BER protein expression and overall BER capacity may help predict sensitivity to temozolomide.Base excision repair (BER) is the predominant pathway for the repair of base damage mediated by endogenous and exogenous stressors (Lindahl and Wood, 1999;Almeida and Sobol, 2007). The repair of DNA bases damaged by alkylation is initiated in mammalian cells by N-methylpurine DNA glycosylase (MPG), also known as alkyladenine DNA glycosylase (Wood et al., 2001). The majority of repair that is initiated by MPG occurs via short-patch BER, a mechanism whereby only one nucleotide is replaced. Once the modified base is removed by MPG, the resulting abasic site is hydrolyzed by AP endonuclease (APE1) (Wood et al., 2001), catalyzing the incision of the damaged strand, leaving a 3ЈOH and a 5Ј-deoxyribose-phosphate moiety (5ЈdRP) at the margins of the repair site. DNA polymerase ␤ (Pol ␤) subsequently hydrolyzes the 5ЈdRP moiety and fills the single nucleotide gap, preparing the strand for ligation by either DNA ligase I or a complex of DNA ligase III␣ and XRCC1.As with many DNA repair processes, BER functions via a series of repair complexes that assemble at the site of the DNA lesion. For the repair of DNA damaged by alkylation, MPG, APE1, Pol ␤, and XRCC1 are essential, with little evidence of effective complementary repair capacity (Almeida and Sobol, 2007). This would suggest that inhibition...

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DNA polymerase beta null mouse embryonic fibroblasts harbor a homozygous null mutation in DNA polymerase iota

Cited by 22 publications

References 60 publications

Negligible impact of pol ι expression on the alkylation sensitivity of pol β-deficient mouse fibroblast cells

Negligible impact of pol ι expression on the alkylation sensitivity of pol β-deficient mouse fibroblast cells

A unified view of base excision repair: Lesion-dependent protein complexes regulated by post-translational modification

Human Methyl Purine DNA Glycosylase and DNA Polymerase β Expression Collectively Predict Sensitivity to Temozolomide

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