Oxidative DNA Damage And Its Processing

Aging

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Abstract

Oxidative lesions are removed from DNA primarily via the base excision repair (BER) pathway. BER is carried out through four enzymatic steps, but is now clear that several other proteins modulate BER efficiency through protein-protein interactions. We and others identified several protein interactions for the core BER enzymes. These protein interactions are physical and functional and together support the "passing of baton" model, in which BER takes place in different steps supported by individual protein interactions that are components of a repair complex, possibly situated at the DNA lesion. Interestingly, these include two proteins associated with premature aging disorders, the Cockayne syndrome (CS) complementation group B gene (CSB) and the Werner syndrome gene (WRN). In CS cells, there are deficiencies in the repair of oxidative DNA damage in the nuclear and mitochondrial DNA, and this may be a major underlying cause of the disease. We found that CSB-deficient cells accumulate oxidized bases, 8-hydroxyguanine and 8-hydroxyadenine, after oxidative stress, consistent with the observation that CSB and oxoguanine DNA glycosylase (OGG1), the major DNA glycosylase for 8-oxoG repair, are in a complex in vivo. We also found that the CSB protein physically interacts with the Nei-like DNA glycosylase, NEIL1, which is also involved in the repair of oxidized bases. This interaction significantly stimulates NEIL1 catalytic activities, both the glycosylase as well as the AP-lyase. The observation that CSB-deficient mice accumulate significantly higher levels of several oxidized DNA bases in brain tissue, including fapyadenine and fapyguanine, supports a role for the CSB protein in the removal of oxidized lesions in vivo. It is notewhorty that Fapy lesions are considered canonical substrates for NEIL1, underscoring the biolgical relevance for this protein interaction.[unreadable] We recently demonstrated that the CSB protein also interacts with PARP1, a protein involved in the early steps of single-strand break repair, and that these two proteins cooperate in the cellular responses to oxidative stress. CSB is a substrate for PARP-1 ribosylation and it is likely that these two proteins function together in the process of base excision. Our results indicate that the CSB protein plays an important role in the repair of oxidative DNA damage and that accumulation of unrepaired lesions, particular in target tissues, like the brain, may be relevant to the CS pathology, which is characterized by severe early onset neurodegeneration.[unreadable] Moreover, we have identified a novel catalytic activity of the CSB protein. Despite having 7 conserved helicase domains (characteristic of the SWI/SNF protein family), the only identified catalytic activity of CSB was ATP hydrolysis. We found that CSB efficiently catalyzes the annealing of two complementary strands of DNA. We are now mapping this novel activity to gain a better understanding of its biological relevance. [unreadable] Repair of 8-oxoG is of special interest since this lesion is believed to be highly mutagenic and accumulates with age. We find that OGG1 interacts with and can be phosphorylated by the cyclin-dependent kinase cdk4. This post-translational modification modulates OGG1 catalytic activity, suggesting a role for signaling pathways in the response to oxidative DNA damage. We are studying other protein interactions of OGG1 in order to understand how repair of oxidative lesions is regulated in vivo. We find that OGG1 also interacts with the recombination protein RAD52, suggesting a possible interplay between these two repair pathways. We find a reciprocal functional interaction between these two proteins, in which RAD52 stimulates OGG1 catalytic activity and OGG1 inhibits RAD52-catalysed DNA strand annealing and invasion. Moreover, the physical interaction between OGG1 and RAD52 increases in cells exposed to oxidative stress, indicating that this interaction is important in the cellular response to oxidative DNA damage.[unreadable] We have recently shown that the WRN protein also interacts physically and functionally with several BER proteins, including polymerase b, flap endonuclease 1 (FEN-1), AP endonuclease (APE) and NEIL-1. We find that WRN strongly stimulates FEN-1 incision, pol b strand displacement activity and NEIL1 glycosylase activity. Further support for a role of WRN in BER comes from our observation that the levels of at least three different oxidative lesions, 8-oxoG, FapyG and FapyA, are significantly elevated in DNA from WRN-deficient cells and that long-patch BER activity is decreased in extracts from cells in which WRN expression is decreased by RNAi. Our results support a model in which WRN promotes long-patch BER by stimulating pol b strand displacement.