Cells from Cockayne syndrome patients and a mouse model of the disease show increased metabolism as a result of impaired autophagy-mediated removal of damaged mitochondria.

We explore the role of DNA damage processing in the progression of cognitive decline by creating a new mouse model. The new model is a cross of a common Alzheimer's disease (AD) mouse (3xTgAD), with a mouse that is heterozygous for the critical DNA base excision repair enzyme, DNA polymerase β. A reduction of this enzyme causes neurodegeneration and aggravates the AD features of the 3xTgAD mouse, inducing neuronal dysfunction, cell death and impairing memory and synaptic plasticity. Transcriptional profiling revealed remarkable similarities in gene expression alterations in brain tissue of human AD patients and 3xTg/Polβ+/− mice including abnormalities suggestive of impaired cellular bioenergetics. Our findings demonstrate that a modest decrement in base excision repair capacity can render the brain more vulnerable to AD-related molecular and cellular alterations.

No abstract

Mitochondrial transcription factor A (TFAM) is an essential component of mitochondrial nucleoids. TFAM plays an important role in mitochondrial transcription and replication. TFAM has been previously reported to inhibit nucleotide excision repair (NER) in vitro but NER has not yet been detected in mitochondria, whereas base excision repair (BER) has been comprehensively characterized in these organelles. The BER proteins are associated with the inner membrane in mitochondria and thus with the mitochondrial nucleoid, where TFAM is also situated. However, a function for TFAM in BER has not yet been investigated. This study examines the role of TFAM in BER. In vitro studies with purified recombinant TFAM indicate that it preferentially binds to DNA containing 8-oxoguanines, but not to abasic sites, uracils, or a gap in the sequence. TFAM inhibited the in vitro incision activity of 8-oxoguanine DNA glycosylase (OGG1), uracil-DNA glycosylase (UDG), apurinic endonuclease 1 (APE1), and nucleotide incorporation by DNA polymerase γ (pol γ). On the other hand, a DNA binding-defective TFAM mutant, L58A, showed less inhibition of BER in vitro. Characterization of TFAM knockdown (KD) cells revealed that these lysates had higher 8oxoG incision activity without changes in αOGG1 protein levels, TFAM KD cells had mild resistance to menadione and increased damage accumulation in the mtDNA when compared to the control cells. In addition, we found that the tumor suppressor p53, which has been shown to interact with and alter the DNA binding activity of TFAM, alleviates TFAM-induced inhibition of BER proteins. Together, the results suggest that TFAM modulates BER in mitochondria by virtue of its DNA binding activity and protein interactions.

Site-specific recombination at the plasmid ColE1 cer site requires the Escherichia coli chromosomal gene xerC. The xerC gene has been localized to the 85-min region of the E. coli chromosome, between cya and uvrD. The nucleotide sequences of the xerC gene and flanking regions have been determined. The xerC gene encodes a protein with a calculated molecular mass of 33.8 kDa. This protein has substantial sequence similarity to the lambda integrase family of site-specific recombinases and is probably the cer recombinase. The xerC gene is expressed as part of a multicistronic unit that includes the dapF gene and two other open reading frames.

Methods for quantifying DNA damage, as well as repair of that damage, in a high-throughput format are lacking. Single cell gel electrophoresis (SCGE; comet assay) is a widely-used method due to its technical simplicity and sensitivity, but the standard comet assay has limitations in reproducibility and throughput. We have advanced the SCGE assay by creating a 96-well hardware platform coupled with dedicated data processing software (CometChip Platform). Based on the original cometchip approach, the CometChip Platform increases capacity ~200 times over the traditional slide-based SCGE protocol, with excellent reproducibility. We tested this platform in several applications, demonstrating a broad range of potential uses including the routine identification of DNA damaging agents, using a 74-compound library provided by the National Toxicology Program. Additionally, we demonstrated how this tool can be used to evaluate human populations by analysis of peripheral blood mononuclear cells to characterize susceptibility to genotoxic exposures, with implications for epidemiological studies. In summary, we demonstrated a high level of reproducibility and quantitative capacity for the CometChip Platform, making it suitable for high-throughput screening to identify and characterize genotoxic agents in large compound libraries, as well as for human epidemiological studies of genetic diversity relating to DNA damage and repair.

SUMMARY RECQL4 is associated with Rothmund-Thomson Syndrome (RTS), a rare autosomal recessive disorder characterized by premature aging, genomic instability and cancer predisposition. RECQL4 is a member of the RecQ-helicase family, and has many similarities to WRN protein, which is also implicated in premature aging. There is no information about whether any of the RecQ helicases play roles in mitochondrial biogenesis, which is strongly implicated in the aging process. Here, we used microscopy to visualize RECQL4 in mitochondria. Fractionation of human and mouse cells also showed that RECQL4 was present in mitochondria. Q-PCR amplification of mitochondrial DNA demonstrated that mtDNA damage accumulated in RECQL4-deficient cells. Microarray analysis suggested that mitochondrial bioenergetic pathways might be affected in RTS. Measurements of mitochondrial bioenergetics showed a reduction in the mitochondrial reserve capacity after lentiviral knockdown of RECQL4 in two different primary cell lines. Additionally, biochemical assays with RECQL4, mitochondrial transcription factor A and mitochondrial DNA polymerase γ showed that the polymerase inhibited RECQL4’s helicase activity. RECQL4 is the first 3′ to 5′ RecQ helicase to be found in both human and mouse mitochondria and the loss of RECQL4 alters mitochondrial integrity.

Ataxia with oculomotor apraxia 1 is caused by mutation in the APTX gene, which encodes the DNA strand-break repair protein aprataxin. Aprataxin exhibits homology to the histidine triad superfamily of nucleotide hydrolases and transferases and removes 5′-adenylate groups from DNA that arise from aborted ligation reactions. We report herein that aprataxin localizes to mitochondria in human cells and we identify an N-terminal amino acid sequence that targets certain isoforms of the protein to this intracellular compartment. We also show that transcripts encoding this unique N-terminal stretch are expressed in the human brain, with highest production in the cerebellum. Depletion of aprataxin in human SH-SY5Y neuroblastoma cells and primary skeletal muscle myoblasts results in mitochondrial dysfunction, which is revealed by reduced citrate synthase activity and mtDNA copy number. Moreover, mtDNA, not nuclear DNA, was found to have higher levels of background DNA damage on aprataxin knockdown, suggesting a direct role for the enzyme in mtDNA processing.