Unilateral ureteral obstruction induces DNA repair by APE1.

Ureteral obstruction is associated with oxidative stress and the development of fibrosis of the kidney parenchyma. Apurinic/apyrimidinic endonuclease (APE1) is an essential DNA repair enzyme for repair of oxidative DNA lesions and regulates several transcription factors. The aim of the present study was to investigate whether APE1 is regulated by acute (24 h) and chronic (7 days) unilateral ureteral obstruction (UUO). APE1 was expressed in essentially all kidney cells with the strongest expression in proximal tubuli. After 24 h of UUO, APE1 mRNA was induced in the cortex, inner stripe of the outer medulla (ISOM), and inner medulla (IM). In contrast, the APE1 protein level was not regulated in the IM and ISOM and only slightly increased in the cortex. APE1 DNA repair activity was not significantly changed. A different pattern of regulation was observed after 7 days of UUO, with an increase of the APE1 mRNA level in the cortex but not in the ISOM and IM. The APE1 protein level in the cortex, ISOM, and IM increased significantly. Importantly, we observed a significant increase in APE1 DNA repair activity in the cortex and IM. To confirm our model, we investigated heme oxygenase-1, collagen type I, fibronectin I, and α-smooth muscle actin levels. In vitro, we found the transcriptional regulatory activity of APE1 to be involved in the upregulation of the profibrotic factor connective tissue growth factor. In summary, APE1 is regulated at different levels after acute and chronic UUO. Thus, our results suggest that DNA repair activity is regulated in response to progressive (7 days) obstruction and that APE1 potentially could play a role in the development of fibrosis in kidney disease.

Cockayne Syndrome group B protein stimulates NEIL2 DNA glycosylase activity.

Cockayne Syndrome is a segmental premature aging syndrome, which can be caused by loss of function of the CSB protein. CSB is essential for genome maintenance and has numerous interaction partners with established roles in different DNA repair pathways including transcription coupled nucleotide excision repair and base excision repair. Here, we describe a new interaction partner for CSB, the DNA glycosylase NEIL2. Using both cell extracts and recombinant proteins, CSB and NEIL2 were found to physically interact independently of DNA. We further found that CSB is able to stimulate NEIL2 glycosylase activity on a 5-hydroxyl uracil lesion in a DNA bubble structure substrate in vitro. A novel 4,6-diamino-5-formamidopyrimidine (FapyA) specific incision activity of NEIL2 was also stimulated by CSB. To further elucidate the biological role of the interaction, immunofluorescence studies were performed, showing an increase in cytoplasmic CSB and NEIL2 co-localization after oxidative stress. Additionally, stalling of the progression of the transcription bubble with α-amanitin resulted in increased co-localization of CSB and NEIL2. Finally, CSB knockdown resulted in reduced incision of 8-hydroxyguanine in a DNA bubble structure using whole cell extracts. Taken together, our data supports a biological role for CSB and NEIL2 in transcription associated base excision repair.

Multiple interaction partners for Cockayne syndrome proteins: implications for genome and transcriptome maintenance.

Cockayne syndrome (CS) is characterized by progressive multisystem degeneration and is classified as a segmental premature aging syndrome. The majority of CS cases are caused by defects in the CS complementation group B (CSB) protein and the rest are mainly caused by defects in the CS complementation group A (CSA) protein. Cells from CS patients are sensitive to UV light and a number of other DNA damaging agents including various types of oxidative stress. The cells also display transcription deficiencies, abnormal apoptotic response to DNA damage, and DNA repair deficiencies. Herein we have critically reviewed the current knowledge about known protein interactions of the CS proteins. The review focuses on the participation of the CSB and CSA proteins in many different protein interactions and complexes, and how these interactions inform us about pathways that are defective in the disease.

Mitochondrial helicases and mitochondrial genome maintenance.

Helicases are essential enzymes that utilize the energy of nucleotide hydrolysis to drive unwinding of nucleic acid duplexes. Helicases play roles in all aspects of DNA metabolism including DNA repair, DNA replication and transcription. The subcellular locations and functions of several helicases have been studied in detail; however, the roles of specific helicases in mitochondrial biology remain poorly characterized. This review presents important recent advances in identifying and characterizing mitochondrial helicases, some of which also operate in the nucleus.

Cockayne syndrome group B protein promotes mitochondrial DNA stability by supporting the DNA repair association with the mitochondrial membrane.

Cockayne syndrome (CS) is a human premature aging disorder associated with severe developmental deficiencies and neurodegeneration, and phenotypically it resembles some mitochondrial DNA (mtDNA) diseases. Most patients belong to complementation group B, and the CS group B (CSB) protein plays a role in genomic maintenance and transcriptome regulation. By immunocytochemistry, mitochondrial fractionation, and Western blotting, we demonstrate that CSB localizes to mitochondria in different types of cells, with increased mitochondrial distribution following menadione-induced oxidative stress. Moreover, our results suggest that CSB plays a significant role in mitochondrial base excision repair (BER) regulation. In particular, we find reduced 8-oxo-guanine, uracil, and 5-hydroxy-uracil BER incision activities in CSB-deficient cells compared to wild-type cells. This deficiency correlates with deficient association of the BER activities with the mitochondrial inner membrane, suggesting that CSB may participate in the anchoring of the DNA repair complex. Increased mutation frequency in mtDNA of CSB-deficient cells demonstrates functional significance of the presence of CSB in the mitochondria. The results in total suggest that CSB plays a direct role in mitochondrial BER by helping recruit, stabilize, and/or retain BER proteins in repair complexes associated with the inner mitochondrial membrane, perhaps providing a novel basis for understanding the complex phenotype of this debilitating disorder.