of work: Living organisms are constantly exposed to oxidative stress from environmental agents and from endogenous metabolic processes. The resulting oxidative modifications occur in proteins, lipids and DNA. Since proteins and lipids are readily degraded and resynthesized, the most significant consequence of oxidative stress are thought to be the DNA modifications, which can lead to the formation of mutations and other types of genomic instability. Many different DNA base changes are formed after oxidative stress. High levels of these lesions are strongly associated with the development of cancer and implicated in the process of aging. Several studies have documented that oxidative DNA lesions accumulate with aging, and it appears that the major site of this accumulation is the mitochondrial DNA rather than the nuclear DNA. Oxidative lesions are removed from DNA primarily by the base excision repair (BER) pathways. 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 accumulated higher levels of two oxidized bases, 8-hydroxyguanine and 8-hydroxyadenine, after oxidative stress and this could be complemented by expression of wild-type CSB. Consistently, we find that CSB and oxoguanine DNA glycosylase (OGG1), the major DNA glycosylase for 8-oxoG repair, are in a complex in vivo, although the two proteins do not interact directly. We recently demonstrated that the CSB protein 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. 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. Repair of 8-oxoG is of special interest since this modification causes mutations, if left unrepaired, 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 have recently shown that the WRN protein also interacts physically and functionally with several BER proteins, including polymerase a, flap endonuclease 1 (FEN-1), AP endonuclease (APE) and NEIL-1. We find that WRN strongly stimulates FEN-1 incision and pol a strand displacement activity and thus promotes long-patch BER. Consistent with an important role for WRN in this process, cells depleted of WRN (via siRNA expression) have decreased long-patch BER activity and accumulate higher levels of oxidative DNA damage.
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