Mammalian base excision repair and DNA polymerase beta

OBJECTIVE: To describe six dogs with congenital abnormalities involving the portal vein, caudal vena cava, or both. ANIMALS: Six client-owned dogs with congenital interruption of the portal vein or the caudal vena cava, or both. METHODS: Portal vein and caudal vena cava anatomy was evaluated by contrast radiography and visualization at surgery. Vascular casts or plastinated specimens were obtained in three animals. RESULTS: Portal blood shunted into the caudal vena cava in four dogs and the left hepatic vein in one. Two of these five dogs also had interruption of the caudal vena cava with continuation as azygous vein, as did an additional dog, in which the portal vein was normally formed. Portal vein interruption was present in 5 of 74 (6.8%) dogs with congenital portosystemic shunts evaluated at the Veterinary Teaching Hospital during the study period. CONCLUSIONS: Serious malformations of the abdominal veins were present in more than 1 in 20 dogs with single congenital portosystemic shunts. CLINICAL RELEVANCE: Veterinarians involved in diagnosis and surgery for portosystemic shunts should be aware of these potential malformations, and portal vein continuity should be evaluated in all dogs before attempting shunt attenuation.     

Chemical approaches toward understanding base excision DNA repair

Despite the importance of DNA repair in protecting the genome, the molecular basis for damage recognition and repair remains poorly understood. In the base excision repair pathway (BER), DNA glycosylases recognize and excise damaged bases from DNA. This review focuses on the recent development of chemical approaches that have been applied to the study of BER enzymes. Several distinctive classes of noncleavable substrate analogs that form stable complexes with DNA glycosylases have recently been designed and synthesized. These analogs have been used for biochemical and structural analyses of protein-DNA complexes involving DNA glycosylases, and for the isolation of a novel DNA glycosylase. An approach to trap covalently a DNA glycosylase-intermediate complex has also been used to elucidate the mechanism of DNA glycosylases.     

Repair of DNA lesions induced by polycyclic aromatic hydrocarbons in human cell-free extracts: involvement of two excision repair mechanisms in vitro

Isotretinoin (ITR), a teratogen in many species, is associated with increased oxidative stress. Metallothionein (MT) is an important tissue antioxidant whose concentrations are induced by zinc. To study the role of supplemental Zn as an inducer of embryonic MT, we injected pregnant CD-1 mice subcutaneously with saline vehicle, or 20 or 40 mg/kg Zn on gestational day (GD) 6.5. After 48 h, embryonic MT concentrations increased in a dose-related manner (r = 0.64, P < 0.05) with Zn treatment. The possible protective role of Zn pretreatment against ITR teratogenicity was investigated in vivo and in vitro. CD-1 mice were pretreated with saline or Zn (20 and 40 mg/kg) on GD 8.5 and 9.5. ITR was administered to both groups of mice via three intragastric intubations of 100 mg ITR/kg at 4 h intervals on GD 10.5. On GD 18.5, Zn pre-treated mice demonstrated decreased ITR-mediated growth retardation, cleft palates and postpartum mortality. A reduction in embryonic MT concentrations was observed in mice exposed to ITR. Mouse embryos cultured on GD 8.5 with an addition of 15 micromol/L Zn for 48 h had a sixfold greater MT concentration (688 microg/g protein) than controls. The Zn pretreatment of cultured embryos prevented malformations and lessened growth retardation caused by 24 h exposure to 17 micromol/L ITR. These results suggest that Zn-mediated induction of MT in mouse embryos could protect against ITR teratogenicity.     

Mammalian base excision repair by DNA polymerases delta and epsilon

Two distinct pathways for completion of base excision repair (BER) have been discovered in eukaryotes: the DNA polymerase beta (Pol beta)-dependent short-patch pathway that involves the replacement of a single nucleotide and the long-patch pathway that entails the resynthesis of 2-6 nucleotides and requires PCNA. We have used cell extracts from Pol beta-deleted mouse fibroblasts to separate subfractions containing either Pol delta or Pol epsilon. These fractions were then tested for their ability to perform both short- and long-patch BER in an in vitro repair assay, using a circular DNA template, containing a single abasic site at a defined position. Remarkably, both Pol delta and Pol epsilon were able to replace a single nucleotide at the lesion site, but the repair reaction is delayed compared to single nucleotide replacement by Pol beta. Furthermore, our observations indicated, that either Pol delta and/or Pol epsilon participate in the long-patch BER. PCNA and RF-C, but not RP-A are required for this process. Our data show for the first time that Pol delta and/or Pol epsilon are directly involved in the long-patch BER of abasic sites and might function as back-up system for Pol beta in one-gap filling reactions.     

Proliferating cell nuclear antigen facilitates excision in long-patch base excision repair

There are two distinct pathways for the removal of modified DNA bases through base excision repair (BER) in vertebrates. Following 5' incision by AP endonuclease, the pathways diverge as two different excision mechanisms are possible. In short-patch repair, DNA polymerase beta accounts for both excision activity and single nucleotide repair synthesis. In long-patch repair, the damage-containing strand is excised by the structure-specific endonuclease FEN-1 and approximately 2-8 nucleotides are incorporated by proliferating cell nuclear antigen (PCNA)-dependent synthesis. PCNA is an accessory factor of DNA polymerases delta and epsilon that is required for DNA replication and repair. PCNA binds to FEN-1 and stimulates its nuclease activity, but the physiological significance of this interaction is unknown. The importance of the PCNA-FEN-1 interaction in BER was investigated. In a reconstituted BER assay system containing FEN-1, omission of PCNA caused the accumulation of pre-excision reaction intermediates which could be converted to completely repaired product by addition of PCNA. When dNTPs were omitted from the reaction to suppress repair synthesis, PCNA was required for the formation of excised reaction intermediates. In contrast, a PCNA mutant that could not bind to FEN-1 was unable to stimulate excision. To further study this effect, a mutant of FEN-1 was identified that retained full nuclease activity but was specifically defective in binding to PCNA. The mutant FEN-1 exhibited one-tenth the specific activity of wild type FEN-1 in the reconstituted BER assay, and this repair defect was due to a kinetic block at the excision step as evidenced by the accumulation of pre-excision intermediates when dNTPs were omitted. These results indicate that PCNA facilitates excision during long-patch BER through its interaction with FEN-1.     

DNA end-joining catalyzed by human cell-free extracts

Mammalian cells defective in DNA end-joining are highly sensitive to ionizing radiation and are immunodeficient because of a failure to complete V(D)J recombination. By using cell-free extracts prepared from human lymphoblastoid cell lines, an in vitro system for end-joining has been developed. Intermolecular ligation was found to be accurate and to depend on DNA ligase IV/Xrcc4 and requires Ku70, Ku86, and DNA-PKcs, the three subunits of the DNA-activated protein kinase DNA-PK. Because these activities are involved in the cellular resistance to x-irradiation and V(D)J recombination, the development of this in vitro system provides an important advance in the study of the mechanism of DNA end-joining in human cells.     

Nucleotide excision repair 3' endonuclease XPG stimulates the activity of base excision repairenzyme thymine glycol DNA glycosylase

An ionizing radiation-induced DNA lesion, thymine glycol, is removed from DNA by a thymine glycol DNA glycosylase with an apurinic/apyrimidinic (AP) lyase activity encoded by the Escherichia coli endonuclease III ( nth ) gene and its homolog in humans. Cells from Cockayne syndrome patients with mutations in the XPG gene show approximately 2-fold reduced global repair of thymine glycol. Hence, I decided to investigate the molecular mechanism of the effect of XPG protein observed in vivo on thymine glycol removal by studying the interactions of XPG protein and human endonuclease III (HsNTH) protein in vitro and the effect of XPG protein on the activity of HsNTH protein on a substrate containing thymine glycol. The XPG protein stimulates the binding of HsNTH protein to its substrate and increases its glycosylase/AP lyase activity by a factor of approximately 2 through direct interaction between the two proteins. These results provide in vitro evidence for a second function of XPG protein in DNA repair and a mechanistic basis for its stimulatory activity on HsNTH protein.     

Different DNA polymerases are involved in the short- and long-patch base excision repair in mammalian cells

Mammalian cells possess two distinct pathways for completion of base excision repair (BER): the DNA polymerase beta (Pol beta)-dependent short-patch pathway (replacement of one nucleotide), which is the main route, and the long-patch pathway (resynthesis of 2-6 nucleotides), which is PCNA-dependent. To address the issue of how these two pathways share their role in BER the ability of Pol beta-defective mammalian cell extracts to repair a single abasic site constructed in a circular duplex plasmid molecule was tested in a standard in vitro repair reaction. Pol beta-deficient extracts were able to perform both BER pathways. However, in the case of the short-patch BER, the repair kinetics was significantly slower than with Pol beta-proficient extracts, while the efficiency of the long-patch synthesis was unaffected by the loss of Pol beta. The repair synthesis was fully dependent on PCNA for the replacement of long patches. These data give the first evidence that in cell extracts DNA polymerases other than Pol beta are specifically involved in the long-patch BER. These DNA polymerases are also able to perform short-patch BER in the absence of PCNA, although less efficiently than Pol beta. These findings lead to a novel model whereby the two BER pathways are characterized by different protein requirements, and a functional redundancy at the level of DNA polymerases provides cells with backup systems.     

Adjacent sequences influence DNA repair accompanying transposon excision in maize

Mobile elements transposing via DNA intermediates often leave small rearrangements, or "transposon footprints," at sites where they excise. Each excision event leaves its own footprint and, at any given site, these vary in size and sequence. Footprint formation involves DNA repair of sequences flanking the element. We have analyzed the footprints formed by a 2-kb Ds element excising from six different sites in exons of the maize waxy (Wx) gene. We find that groups of footprints left at individual sites are surprisingly nonrandom; different excision products predominate consistently at each site. Less frequent footprints left by each insertion appear related to the predominant type. The data suggest that flanking sequences affect the DNA repair processes associated with element excision. Two models have been proposed to explain footprint formation, one featuring a 5' exonuclease and the other featuring hairpin loop formation and an endonuclease. Our data have interesting implications for both these models. Evidence is also presented to support the presence of a separate excision mechanism that can remove Ac/Ds elements without leaving any footprint and that operates in parallel with the footprint-forming mechanism.     

Proteins that participate in nucleotide excision repair of DNA in mammalian cells

The most versatile strategy for repair of damage to DNA, and the main process for repair of UV-induced damage, is nucleotide excision repair. In mammalian cells, the complete mechanism involves more than 20 polypeptides, and defects in many of these are associated with various forms of inherited disorders in humans. The syndrome xeroderma pigmentosum (XP) is associated with mutagen hypersensitivity and increased cancer frequency, and studies of the nucleotide excision repair defect in this disease have been particularly informative. Many of the XP proteins are now being characterized. XPA binds to DNA, with a preference for damaged base pairs. XPC activity is part of a protein complex with single-stranded DNA binding activity. The XPG protein is a nuclease.     

The fission yeast UVDR DNA repair pathway is inducible

In addition to nucleotide excision repair (NER), the fission yeast Schizosaccharomyces pombe possesses a UV damage endonuclease (UVDE) for the excision of cyclobutane pyrimidine dimers and 6-4 pyrimidine pyrimidones. We have previously described UVDE as part of an alternative excision repair pathway, UVDR, for UV damage repair. The existence of two excision repair processes has long been postulated to exist in S.pombe, as NER-deficient mutants are still proficient in the excision of UV photoproducts. UVDE recognizes the phosphodiester bond immediately 5'of the UV photoproducts as the initiating event in this process. We show here that UVDE activity is inducible at both the level of uve1+ mRNA and UVDE enzyme activity. Further, we show that UVDE activity is regulated by the product of the rad12 gene.     

Base excision repair deficient mice lacking the Aag alkyladenine DNA glycosylase

3-methyladenine (3MeA) DNA glycosylases remove 3MeAs from alkylated DNA to initiate the base excision repair pathway. Here we report the generation of mice deficient in the 3MeA DNA glycosylase encoded by the Aag (Mpg) gene. Alkyladenine DNA glycosylase turns out to be the major DNA glycosylase not only for the cytotoxic 3MeA DNA lesion, but also for the mutagenic 1,N6-ethenoadenine (epsilonA) and hypoxanthine lesions. Aag appears to be the only 3MeA and hypoxanthine DNA glycosylase in liver, testes, kidney, and lung, and the only epsilonA DNA glycosylase in liver, testes, and kidney; another epsilonA DNA glycosylase may be expressed in lung. Although alkyladenine DNA glycosylase has the capacity to remove 8-oxoguanine DNA lesions, it does not appear to be the major glycosylase for 8-oxoguanine repair. Fibroblasts derived from Aag -/- mice are alkylation sensitive, indicating that Aag -/- mice may be similarly sensitive.     

Destabilization of tracts of simple repetitive DNA in yeast by mutations affecting DNA mismatch repair

The genomes of all eukaryotes contain tracts of DNA in which a single base or a small number of bases is repeated. Expansions of such tracts have been associated with several human disorders including the fragile X syndrome. In addition, simple repeats are unstable in certain forms of colorectal cancer, suggesting a defect in DNA replication or repair. We show here that mutations in any three yeast genes involved in DNA mismatch repair (PMS1, MLH1 and MSH2) lead to 100- to 700-fold increases in tract instability, whereas mutations that eliminate the proof-reading function of DNA polymerases have little effect. The meiotic stability of the tracts is similar to the mitotic stability. These results suggest that tract instability is associated with DNA polymerases slipping during replication, and that some types of colorectal cancer may reflect mutations in genes involved in DNA mismatch repair.     

Triplet repeats form secondary structures that escape DNA repair in yeast

Several human neurodegenerative diseases result from expansion of CTG/CAG or CGG/CCG triplet repeats. The finding that single-stranded CNG repeats form hairpin-like structures in vitro has led to the hypothesis that DNA secondary structure formation is an important component of the expansion mechanism. We show that single-stranded DNA loops containing 10 CTG/CAG or CGG/CCG repeats are inefficiently repaired during meiotic recombination in Saccharomyces cerevisiae. Comparisons of the repair of DNA loops with palindromic and nonpalindromic sequences suggest that this inefficient repair reflects the ability of these sequences to form hairpin structures in vivo.     

Two distinct DNA ligase activities in mitotic extracts of the yeast Saccharomyces cerevisiae

Four biochemically distinct DNA ligases have been identified in mammalian cells. One of these enzymes, DNA ligase I, is functionally homologous to the DNA ligase encoded by the Saccharomyces cerevisiae CDC9 gene. Cdc9 DNA ligase has been assumed to be the only species of DNA ligase in this organism. In the present study we have identified a second DNA ligase activity in mitotic extracts of S. cerevisiae with chromatographic properties different from Cdc9 DNA ligase, which is the major DNA joining activity. This minor DNA joining activity, which contributes 5-10% of the total cellular DNA joining activity, forms a 90 kDa enzyme-adenylate intermediate which, unlike the Cdc9 enzyme-adenylate intermediate, reacts with an oligo (pdT)/poly (rA) substrate. The levels of the minor DNA joining activity are not altered by mutation or by overexpression of the CDC9 gene. Furthermore, the 90 kDa polypeptide is not recognized by a Cdc9 antiserum. Since this minor species does not appear to be a modified form of Cdc9 DNA ligase, it has been designated as S. cerevisiae DNA ligase II. Based on the similarities in polynucleotide substrate specificity, this enzyme may be the functional homolog of mammalian DNA ligase III or IV.     

Endogenous lesions, S-phase-independent spontaneous mutations, and evolutionary strategies for base excision repair

The mitogen-activated protein (MAP) kinases (p44mapk and p42mapk), also known as extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2), are activated in response to a variety of extracellular signals, including growth factors, hormones and, neurotransmitters. We have investigated MAP kinase signal transduction pathways in normal human osteoblastic cells. Normal human bone marrow stromal (HBMS), osteoblastic (HOB), and human (TE85, MG-63, SaOS-2), rat (ROS 17/2.8, UMR-106) and mouse (MC3T3-E1) osteoblastic cell lines contained immunodetectable p44mapk/ERK1 and p42mapk/ERK2. MAP kinase activity was measured by 'in-gel' assay using myelin basic protein as the substrate. Mainly ERK2 was rapidly activated (within 10 min) by bFGF, IGF-I and PDGF-BB in normal HOB, HBMS and human osteosarcoma cells, whereas both ERK1 and ERK2 were activated by growth factors in rat osteoblast-like cell lines, ROS 17/2.8 and UMR-106. The ERK1 activation was greater than the ERK2 in ROS 17/2.8 cells. Furthermore, ERK2 was also activated by bFGF and PDGF-BB in the mouse osteoblastic cell line, MC3T3-E1. This is the first demonstration of inter-species differences in the activation of MAP kinases in osteoblastic cells. Cyclic AMP derivatives or cAMP generating agents such as PTH and forskolin inhibited ERK2 activation by bFGF and PDGF-BB suggesting a 'cross-talk' between the two different signalling pathways activated by receptor tyrosine kinases and cAMP-dependent protein kinase. The accumulated results also suggest that the MAP kinases may be involved in mediating mitogenic and other biological actions of bFGF, IGF-I and PDGF-BB in normal human osteoblastic and bone marrow stromal cells.