Substrate channeling in mammalian base excision repair pathways: passing the baton

The current model for base excision repair (BER) involves two general sub-pathways termed single-nucleotide BER and long patch BER that are distinguished by their repair patch sizes and the enzymes/co-factors involved. Both sub-pathways involve a series of sequential steps from initiation to completion of repair. The BER sub-pathways are designed to sequester the various intermediates, passing them along from one step to the next without allowing these toxic molecules to trigger cell cycle arrest, necrotic cell death, or apoptosis. Although a variety of DNA-protein and protein-protein interactions are known for the BER intermediates and enzymes/co-factors, the molecular mechanisms accounting for step-to-step coordination are not well understood. In the present study we designed an in vitro assay to explore the question of whether there is a channeling or "hand-off" of the repair intermediates during BER in vitro. The results show that when BER enzymes are pre-bound to the initial single-nucleotide BER intermediate, the DNA is channeled from apurinic/apyrimidinic endonuclease 1 to DNA polymerase β and then to DNA ligase. In the long patch BER subpathway, where the 5'-end of the incised strand is blocked, the intermediate after DNA polymerase β gap filling is not channeled to the subsequent enzyme, flap endonuclease 1. Instead, flap endonuclease 1 must recognize and bind to the intermediate in competition with other molecules.     

Activation of ras signaling pathway by 8-oxoguanine DNA glycosylase bound to its excision product, 8-oxoguanine

8-Oxo-7,8-dihydroguanine (8-oxoG), arguably the most abundant base lesion induced in mammalian genomes by reactive oxygen species, is repaired via the base excision repair pathway that is initiated with the excision of 8-oxoG by OGG1. Here we show that OGG1 binds the 8-oxoG base with high affinity and that the complex then interacts with canonical Ras family GTPases to catalyze replacement of GDP with GTP, thus serving as a guanine nuclear exchange factor. OGG1-mediated activation of Ras leads to phosphorylation of the mitogen-activated kinases MEK1,2/ERK1,2 and increasing downstream gene expression. These studies document for the first time that in addition to its role in repairing oxidized purines, OGG1 has an independent guanine nuclear exchange factor activity when bound to 8-oxoG.     

Influence of Oxidized Purine Processing on Strand Directionality of Mismatch Repair

Replicative DNA polymerases are high fidelity enzymes that misincorporate nucleotides into nascent DNA with a frequency lower than [1/10⁵], and this precision is improved to about [1/10⁷] by their proofreading activity. Because this fidelity is insufficient to replicate most genomes without error, nature evolved postreplicative mismatch repair (MMR), which improves the fidelity of DNA replication by up to 3 orders of magnitude through correcting biosynthetic errors that escaped proofreading. MMR must be able to recognize non-Watson-Crick base pairs and excise the misincorporated nucleotides from the nascent DNA strand, which carries by definition the erroneous genetic information. In eukaryotes, MMR is believed to be directed to the nascent strand by preexisting discontinuities such as gaps between Okazaki fragments in the lagging strand or breaks in the leading strand generated by the mismatch-activated endonuclease of the MutL homologs PMS1 in yeast and PMS2 in vertebrates. We recently demonstrated that the eukaryotic MMR machinery can make use also of strand breaks arising during excision of uracils or ribonucleotides from DNA. We now show that intermediates of MutY homolog-dependent excision of adenines mispaired with 8-oxoguanine (G^O) also act as MMR initiation sites in extracts of human cells or Xenopus laevis eggs. Unexpectedly, G^O/C pairs were not processed in these extracts and failed to affect MMR directionality, but extracts supplemented with exogenous 8-oxoguanine DNA glycosylase (OGG1) did so. Because OGG1-mediated excision of G^O might misdirect MMR to the template strand, our findings suggest that OGG1 activity might be inhibited during MMR.     

Apoptosis Induced by Persistent Single-strand Breaks in Mitochondrial Genome: CRITICAL ROLE OF EXOG (5′-EXO/ENDONUCLEASE) IN THEIR REPAIR*

Reactive oxygen species (ROS), continuously generated as by-products of respiration, inflict more damage on the mitochondrial (mt) than on the nuclear genome because of the nonchromatinized nature and proximity to the ROS source of the mitochondrial genome. Such damage, particularly single-strand breaks (SSBs) with 5′-blocking deoxyribose products generated directly or as repair intermediates for oxidized bases, is repaired via the base excision/SSB repair pathway in both nuclear and mt genomes. Here, we show that EXOG, a 5′-exo/endonuclease and unique to the mitochondria unlike FEN1 or DNA2, which, like EXOG, has been implicated in the removal of the 5′-blocking residue, is required for repairing endogenous SSBs in the mt genome. EXOG depletion induces persistent SSBs in the mtDNA, enhances ROS levels, and causes apoptosis in normal cells but not in mt genome-deficient rho0 cells. Thus, these data show for the first time that persistent SSBs in the mt genome alone could provide the initial trigger for apoptotic signaling in mammalian cells.     

Specific Inhibition of NEIL-initiated Repair of Oxidized Base Damage in Human Genome by Copper and Iron: POTENTIAL ETIOLOGICAL LINKAGE TO NEURODEGENERATIVE DISEASES*

Dyshomeostasis of transition metals iron and copper as well as accumulation of oxidative DNA damage have been implicated in multitude of human neurodegenerative diseases, including Alzheimer disease and Parkinson disease. These metals oxidize DNA bases by generating reactive oxygen species. Most oxidized bases in mammalian genomes are repaired via the base excision repair pathway, initiated with one of four major DNA glycosylases: NTH1 or OGG1 (of the Nth family) or NEIL1 or NEIL2 (of the Nei family). Here we show that Fe(II/III) and Cu(II) at physiological levels bind to NEIL1 and NEIL2 to alter their secondary structure and strongly inhibit repair of mutagenic 5-hydroxyuracil, a common cytosine oxidation product, both in vitro and in neuroblastoma (SH-SY5Y) cell extract by affecting the base excision and AP lyase activities of NEILs. The specificity of iron/copper inhibition of NEILs is indicated by a lack of similar inhibition of OGG1, which also indicated that the inhibition is due to metal binding to the enzymes and not DNA. Fluorescence and surface plasmon resonance studies show submicromolar binding of copper/iron to NEILs but not OGG1. Furthermore, Fe(II) inhibits the interaction of NEIL1 with downstream base excision repair proteins DNA polymerase β and flap endonuclease-1 by 4–6-fold. These results indicate that iron/copper overload in the neurodegenerative diseases could act as a double-edged sword by both increasing oxidative genome damage and preventing their repair. Interestingly, specific chelators, including the natural chemopreventive compound curcumin, reverse the inhibition of NEILs both in vitro and in cells, suggesting their therapeutic potential.     

Novel role of base excision repair in mediating cisplatin cytotoxicity

Using isogenic mouse embryonic fibroblasts and human cancer cell lines, we show that cells defective in base excision repair (BER) display a cisplatin-specific resistant phenotype. This was accompanied by enhanced repair of cisplatin interstrand cross-links (ICLs) and ICL-induced DNA double strand breaks, but not intrastrand adducts. Cisplatin induces abasic sites with a reduced accumulation in uracil DNA glycosylase (UNG) null cells. We show that cytosines that flank the cisplatin ICLs undergo preferential oxidative deamination in vitro, and AP endonuclease 1 (APE1) can cleave the resulting ICL DNA substrate following removal of the flanking uracil. We also show that DNA polymerase β has low fidelity at the cisplatin ICL site after APE1 incision. Down-regulating ERCC1-XPF in BER-deficient cells restored cisplatin sensitivity. Based on our results, we propose a novel model in which BER plays a positive role in maintaining cisplatin cytotoxicity by competing with the productive cisplatin ICL DNA repair pathways.     

NEIL2-initiated, APE-independent repair of oxidized bases in DNA: Evidence for a repair complex in human cells

DNA glycosylases/AP lyases initiate repair of oxidized bases in the genomes of all organisms by excising these lesions and then cleaving the DNA strand at the resulting abasic (AP) sites and generate 3' phospho alpha,beta-unsaturated aldehyde (3' PUA) or 3' phosphate (3' P) terminus. In Escherichia coli, the AP-endonucleases (APEs) hydrolyze both 3' blocking groups (3' PUA and 3' P) to generate the 3'-OH termini needed for repair synthesis. In mammalian cells, the previously characterized DNA glycosylases, NTH1 and OGG1, produce 3' PUA, which is removed by the only AP-endonuclease, APE1. However, APE1 is barely active in removing 3' phosphate generated by the recently discovered mammalian DNA glycosylases NEIL1 and NEIL2. We showed earlier that the 3' phosphate generated by NEIL1 is efficiently removed by polynucleotide kinase (PNK) and not APE1. Here we show that the NEIL2-initiated repair of 5-hydroxyuracil (5-OHU) similarly requires PNK. We have also observed stable interaction between NEIL2 and other BER proteins DNA polymerase beta (Pol beta), DNA ligase IIIalpha (Lig IIIalpha) and XRCC1. In spite of their limited sequence homology, NEIL1 and NEIL2 interact with the same domains of Pol beta and Lig IIIalpha. Surprisingly, while the catalytically dispensable C-terminal region of NEIL1 is the common interacting domain, the essential N-terminal segment of NEIL2 is involved in analogous interaction. The BER proteins including NEIL2, PNK, Pol beta, Lig IIIalpha and XRCC1 (but not APE1) could be isolated as a complex from human cells, competent for repair of 5-OHU in plasmid DNA.     

DNA Sequence Context Effects on the Glycosylase Activity of Human 8-Oxoguanine DNA Glycosylase

Human 8-oxoguanine DNA glycosylase (OGG1) is a key enzyme involved in removing 7,8-dihydro-8-oxoguanine (8-oxoG), a highly mutagenic DNA lesion generated by oxidative stress. The removal of 8-oxoG by OGG1 is affected by the local DNA sequence, and this feature most likely contributes to observed mutational hot spots in genomic DNA. To elucidate the influence of local DNA sequence on 8-oxoG excision activity of OGG1, we conducted steady-state, pre-steady-state, and single turnover kinetic evaluation of OGG1 in alternate DNA sequence contexts. The sequence context effect was studied for a mutational hot spot at a CpG dinucleotide. Altering either the global DNA sequence or the 5′-flanking unmodified base pair failed to influence the excision of 8-oxoG. Methylation of the cytosine 5′ to 8-oxoG also did not affect 8-oxoG excision. In contrast, a 5′-neighboring mismatch strongly decreased the rate of 8-oxoG base removal. Substituting the 5′-C in the CpG dinucleotide with T, A, or tetrahydrofuran (i.e. T:G, A:G, and tetrahydrofuran:G mispairs) resulted in a 10-, 13-, and 4-fold decrease in the rate constant for 8-oxoG excision, respectively. A greater loss in activity was observed when T:C or A:C was positioned 5′ of 8-oxoG (59- and 108-fold, respectively). These results indicate that neighboring structural abnormalities 5′ to 8-oxoG deter its repair thereby enhancing its mutagenic potential.     

Cancer cell survival following DNA damage-mediated premature senescence is regulated by mammalian target of rapamycin (mTOR)-dependent Inhibition of sirtuin 1

DNA-damaging agents can induce premature senescence in cancer cells, which contributes to the static effects of cancer. However, senescent cancer cells may re-enter the cell cycle and lead to tumor relapse. Understanding the mechanisms that control the viability of senescent cells may be helpful in eliminating these cells before they can regrow. Treating human squamous cell carcinoma (SCC) cells with the anti-cancer compounds, resveratrol and doxorubicin, triggered p53-independent premature senescence by invoking oxidative stress-mediated DNA damage. This process involved the mTOR-dependent phosphorylation of SIRT1 at serine 47, resulting in the inhibition of the deacetylase activity of SIRT1. SIRT1 phosphorylation caused concomitant increases in p65/RelA NF-κB acetylation and the expression of an anti-apoptotic Bfl-1/A1. SIRT1 physically interacts with the mTOR-Raptor complex, and a single amino acid substitution in the TOS (TOR signaling) motif in the SIRT1 prevented Ser-47 phosphorylation and Bfl-1/A1 induction. The pharmacologic and genetic inhibition of mTOR, unphosphorylatable S47A, or F474A TOS mutants restored SIRT1 deacetylase activity, blocked Bfl-1/A1 induction, and sensitized prematurely senescent SCC cells for apoptosis. We further show that the treatment of UVB-induced SCCs with doxorubicin transiently stabilized tumor growth but was followed by tumor regrowth upon drug removal in p53(+/-)/SKH-1 mice. The subsequent treatment of stabilized SCCs with rapamycin decreased tumor size and induced caspase-3 activation. These results demonstrate that the inhibition of SIRT1 by mTOR fosters survival of DNA damage-induced prematurely senescent SCC cells via Bfl-1/A1 in the absence of functional p53.     

Radicals in Berkeley?

In a previous autobiographical sketch for DNA Repair (Linn, S. (2012) Life in the serendipitous lane: excitement and gratification in studying DNA repair. DNA Repair 11, 595–605), I wrote about my involvement in research on mechanisms of DNA repair. In this Reflections, I look back at how I became interested in free radical chemistry and biology and outline some of our bizarre (at the time) observations. Of course, these studies could never have succeeded without the exceptional aid of my mentors: my teachers; the undergraduate and graduate students, postdoctoral fellows, and senior lab visitors in my laboratory; and my faculty and staff colleagues here at Berkeley. I am so indebted to each and every one of these individuals for their efforts to overcome my ignorance and set me on the straight and narrow path to success in research. I regret that I cannot mention and thank each of these mentors individually.     

Selenium Compounds Activate ATM-dependent DNA Damage Response via the Mismatch Repair Protein hMLH1 in Colorectal Cancer Cells

Epidemiological and animal studies indicate that selenium supplementation suppresses risk of colorectal and other cancers. The majority of colorectal cancers are characterized by a defective DNA mismatch repair (MMR). Here, we have employed the MMR-deficient HCT 116 colorectal cancer cells and the MMR-proficient HCT 116 cells with hMLH1 complementation to investigate the role of hMLH1 in selenium-induced DNA damage response, a tumorigenesis barrier. The ATM (ataxia telangiectasia mutated) protein responds to clastogens and initiates DNA damage response. We show that hMLH1 complementation sensitizes HCT 116 cells to methylseleninic acid, methylselenocysteine, and sodium selenite via reactive oxygen species and facilitates the selenium-induced oxidative 8-oxoguanine damage, DNA breaks, G₂/M checkpoint response, and ATM pathway activation. Pretreatment of the hMLH1-complemented HCT 116 cells with the antioxidant N-acetylcysteine or 2,2,6,6-tetramethylpiperidine-1-oxyl or the ATM kinase inhibitor KU55933 suppresses hMLH1-dependent DNA damage response to selenium exposure. Selenium treatment stimulates the association between hMLH1 and hPMS2 proteins, a heterodimer critical for functional MMR, in a manner dependent on ATM and reactive oxygen species. Taken together, the results suggest a new role of selenium in mitigating tumorigenesis by targeting the MMR pathway, whereby the lack of hMLH1 renders the HCT 116 colorectal cancer cells resistant to selenium-induced DNA damage response.     

Induction of Cell Death through Alteration of Oxidants and Antioxidants in Lung Epithelial Cells Exposed to High Energy Protons

Radiation affects several cellular and molecular processes, including double strand breakage and modifications of sugar moieties and bases. In outer space, protons are the primary radiation source that poses a range of potential health risks to astronauts. On the other hand, the use of proton irradiation for tumor radiation therapy is increasing, as it largely spares healthy tissues while killing tumor tissues. Although radiation-related research has been conducted extensively, the molecular toxicology and cellular mechanisms affected by proton irradiation remain poorly understood. Therefore, in this study, we irradiated rat lung epithelial cells with different doses of protons and investigated their effects on cell proliferation and death. Our data show an inhibition of cell proliferation in proton-irradiated cells with a significant dose-dependent activation and repression of reactive oxygen species and antioxidants glutathione and superoxide dismutase, respectively, compared with control cells. In addition, the activities of apoptosis-related genes such as caspase-3 and -8 were induced in a dose-dependent manner with corresponding increased levels of DNA fragmentation in proton-irradiated cells compared with control cells. Together, our results show that proton irradiation alters oxidant and antioxidant levels in cells to activate the apoptotic pathway for cell death.     

Germ Line Variants of Human N-Methylpurine DNA Glycosylase Show Impaired DNA Repair Activity and Facilitate 1,N6-Ethenoadenine-induced Mutations

Human N-methylpurine DNA glycosylase (hMPG) initiates base excision repair of a number of structurally diverse purine bases including 1,N⁶-ethenoadenine, hypoxanthine, and alkylation adducts in DNA. Genetic studies discovered at least eight validated non-synonymous single nucleotide polymorphisms (nsSNPs) of the hMPG gene in human populations that result in specific single amino acid substitutions. In this study, we tested the functional consequences of these nsSNPs of hMPG. Our results showed that two specific arginine residues, Arg-141 and Arg-120, are important for the activity of hMPG as the germ line variants R120C and R141Q had reduced enzymatic activity in vitro as well as in mammalian cells. Expression of these two variants in mammalian cells lacking endogenous MPG also showed an increase in mutations and sensitivity to an alkylating agent compared with the WT hMPG. Real time binding experiments by surface plasmon resonance spectroscopy suggested that these variants have substantial reduction in the equilibrium dissociation constant of binding (K_D) of hMPG toward 1,N⁶-ethenoadenine-containing oligonucleotide (ϵA-DNA). Pre-steady-state kinetic studies showed that the substitutions at arginine residues affected the turnover of the enzyme significantly under multiple turnover condition. Surface plasmon resonance spectroscopy further showed that both variants had significantly decreased nonspecific (undamaged) DNA binding. Molecular modeling suggested that R141Q substitution may have resulted in a direct loss of the salt bridge between ϵA-DNA and hMPG, whereas R120C substitution redistributed, at a distance, the interactions among residues in the catalytic pocket. Together our results suggest that individuals carrying R120C and R141Q MPG variants may be at risk for genomic instability and associated diseases as a consequence.