Enhanced repair of pyrimidine dimers in coding and non-coding genomic sequences in CHO cells expressing a prokaryotic DNA repair gene

We have previously demonstrated that the active dihydrofolatereductase (DHFR) gene is efficiently repaired in Chinese hamsterovary (CHO) cells which remove only a small fraction of u.v.-inducedpyrimidine dimers from the overall genome. Preferential DNArepair of essential genes may explain why the u.v. resistanceof normal CHO cells is as high as that of fully repair-proficientnormal human cells. In this report, we have studied the removalof pyrimidine dimers in a CHO cell line expressing the cloneddenV gene from bacteriophage T4 which codes for the pyrimidinedimer specific enzyme T4 endonuclease V (T4 endo V). This cellline was derived from a u.v.-sensitive excision deficient mutantof a CHO wild type line by transformation with the denV gene,and partial restoration of u.v. resistance was achieved. Wehave examined an important aspect of the u.v. excision repairin these denV⁺ cells by studying the repair efficiencies inthe active DHFR gene and in a non-coding sequence located downstreamfrom it. In the u.v.-sensitive CHO mutant cell line from whichthe denV⁺ was derived, we detected no pyrimidine dimer removalfrom the gene or from the downstream sequence after irradiationof the cells with 20 J/m² u.v. (254 nm) light. In the wild typeCHO cells, 50% of the pyrimidine dimers were removed from asequence in the DHFR gene within 8 h, whereas none were removedfrom the downstream sequence in that period. This representsthe normal pattern of preferential DNA repair of active genes,which we have described in previous communications. In the denV⁺cells, 70% of the pyrimidine dimers were removed from both theDHFR gene and from the downstream sequence; these cells thusrepair both coding and non-coding regions of the genome andshow no pattern of preferential repair. The endogenous activitythat initiates excision repair in normal CHO cells is evidentlymuch more restricted in its accessibility to DNA lesions inchromatin than is the activity in cells containing substantialamounts of the small T4 endo V enzyme.     

875 Cases of bacterial meningitis: Diagnostic procedures and the impact of preadmission antibiotic therapy Part III of a three-part series

Data on the bacteriological findings, diagnostic measures and clinical course of 875 patients with bacterial meningitis are presented. Findings from the medical records and from a follow-up questionnaire survey of 667 of these cases revealed no significant difference between patients treated with antibiotics before admission (pretreated) and those who were not treated before admission (non-pretreated) with respect to clinical condition on admission, mortality and late sequelae. Pretreatment was, however, associated with a longer duration of symptoms. Apart from cases due to Neisseria meningitidis, there were no significant differences in diagnostic findings between pretreated and non-pretreated cases. In the group of pretreated meningococcal patients, however, positive blood cultures, pleiocytosis in the cerebrospinal fluid (CSF) and positive cultures from sites other than blood and CSF were less frequent than in the non-pretreated cases.     

Altered spectra of hypermutation in antibodies from mice deficient for the DNA mismatch repair protein PMS2

Mutations are introduced into rearranged Ig variable genes at a frequency of 10⁻² mutations per base pair by an unknown mechanism. Assuming that DNA repair pathways generate or remove mutations, the frequency and pattern of mutation will be different in variable genes from mice defective in repair. Therefore, hypermutation was studied in mice deficient for either the DNA nucleotide excision repair gene Xpa or the mismatch repair gene Pms2. High levels of mutation were found in variable genes from XPA-deficient and PMS2-deficient mice, indicating that neither nucleotide excision repair nor mismatch repair pathways generate hypermutation. However, variable genes from PMS2-deficient mice had significantly more adjacent base substitutions than genes from wild-type or XPA-deficient mice. By using a biochemical assay, we confirmed that tandem mispairs were repaired by wild-type cells but not by Pms2^−/− human or murine cells. The data indicate that tandem substitutions are produced by the hypermutation mechanism and then processed by a PMS2-dependent pathway.     

Fever in pregnancy and offspring head circumference

Purpose

To examine whether maternal fever during pregnancy is associated with reduced head circumference and risk of microcephaly at birth.

Mitochondrial repair of 8-oxoguanine and changes with aging

Reactive oxygen species (ROS) are formed in all living organisms as a by-product of normal metabolism (endogenous sources) and as a consequence of exposure to environmental compounds (exogenous sources). Endogenous ROS are largely formed during oxidative phosphorylation in the mitochondria and, therefore, mitochondrial DNA (mtDNA) is at particularly high risk of ROS-induced damage. Mitochondria are essential for cell viability, and oxidative damage to mtDNA has been implicated as a causative factor in a wide variety of degenerative diseases, and in cancer and aging. One of the most common oxidative DNA lesions is 7,8-dihydro-8-oxoguanine (8-oxoG), which can introduce G/C to T/A transversions after DNA replication. Oxidative DNA base lesions, including 8-oxoG, are repaired primarily by the base excision repair (BER) pathway. While we know much about how this pathway functions in processing the nuclear DNA lesions, little is yet known about BER in mitochondria. We have used a number of different approaches to explore the mechanisms of DNA damage processing in the mtDNA. We have been able to demonstrate that mammalian mitochondria efficiently remove 8-oxoG from their genome, and that the efficiency of 8-oxoG incision increases with age in rats and mice. Yet 8-oxoG accumulates in mtDNA during aging. Changes in mitochondrial function with age have been observed in several organisms and accumulation of DNA lesions in mtDNA with age may be an underlying cause for numerous age-associated diseases including cancer.     

Mitochondrial DNA repair and association with aging – An update

Mitochondrial DNA is constantly exposed to oxidative injury. Due to its location close to the main site of reactive oxygen species, the inner mitochondrial membrane, mtDNA is more susceptible than nuclear DNA to oxidative damage. The accumulation of DNA damage is thought to play a critical role in the aging process and to be particularly deleterious in post-mitotic cells. Thus, DNA repair is an important mechanism for maintenance of genomic integrity. Despite the importance of mitochondria in the aging process, it was thought for many years that mitochondria lacked an enzymatic DNA repair system comparable to that in the nuclear compartment. However, it is now well established that DNA repair actively takes place in mitochondria. Oxidative DNA damage processing, base excision repair mechanisms were the first to be described in these organelles, and consequently the best understood. However, new proteins and novel DNA repair pathways, thought to be exclusively present in the nucleus, have recently been described also to be present in mitochondria. Here we review the main mitochondrial DNA repair pathways and their association with the aging process.