The Fanconi Anemia Pathway in Inflammatory Senescent Cells

National Institute On Aging

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Abstract

We focused on two issues in our approach to this question. The first was concerned with the participation in the DDR of FANCD2, the central protein of the FA pathway. Most of our information about the DDR comes from studies of double strand breaks (DSBs). Some proteins are recruited close to DNA breaks, while others are located hundreds to thousands of bases away. However, DSBs are relatively rare in cultured cells and even less common in circulating lymphocytes. In contrast, DNA base damage and loss occurs at least 3 orders of magnitude more frequently. Consequently, we examined the participation of FANCD2 in the DDR induced by a base reactive compound, to which we attached an immunotag. We developed a new experimental approach, based on the immunotag, to differentiate DDR proteins that were close to the DNA damage from those that were distant. FANCD2 was recruited to the DNA damage in two spatially separable cohorts. One was located in close proximity to the DNA lesion and contributed to the repair of the lesion. A second, somewhat larger fraction, associated with, and was dependent on, proteins located at a distance from the base modification. This cohort had no involvement in removal of the damage. It is likely that this fraction is involved in stress signaling. We are now examining the role it plays in the regulation of inflammatory pathways. We have also identified a new model of cell senescence that differs from others described in recent publications. We expose cells to a DNA damaging agent. These cells become senescent after 7-10 days. In contrast to other cell culture models with senescent cell frequencies of 60-70%, 100% of these cells become senescence. Previous literature indicates an important contribution of the histone variant H2AX towards development of senescence. However, in the new system senescence is independent of H2AX. DNA Damage Response markers are visible in foci in the senescent cells, despite the absence of phospho-H2AX. FANCD2 is a component of the foci during entry into senescence, but disappears as the cells become fully senescent. The basis for the absence is the focus of current study.