Werner's syndrome (WS) is a homozygous recessive disease characterized by early onset of many characteristics of normal aging, such as wrinkling of the skin, graying of the hair, cataracts, diabetes, and osteoporosis. Because of the acceleration of aging in WS, the study of this disease will hopefully shed light on the degenerative processes that occur in normal aging. Cells from WS patients grow more slowly and senescence at an earlier population doubling than age-matched normal cells, possibly because these cells appear to lose the telomeric ends of their chromosomes at an accelerated rate. In general, WS cells have a high level of genomic instability, with increased amounts of DNA deletions, insertions, and rearrangements. These effects could potentially be the result of defects in DNA repair, replication, and/or recombination, although the actual biochemical defect remains unknown. The gene that is defective in WS, the WRN gene, has been identified and characterized. We have made purified WRN protein for use in a number of basic and complex biochemical assays. We are pursuing several avenues to identify and characterize the biochemical defect in WS cells. WRN protein has helicase activity and will unwind small and large DNA duplex constructs. Additionally, WRN has a 3-5'exonuclease function. Recently, we showed that the RQC domain of WRN is critical for its DNA binding and catalytic activities. This work also revealed that mutations in the RQC could impact the exonuclease domain of WRN, which was previously thought to be an autonomous domain. We are comparing WRN to that of the other RecQ helicases that are all involved in the maintenance of genome stability. We continue to use confocal microscopy as a means to investigate the dynamic behavior of WRN and the other RecQ helicases. This technique has revealed that each of the RecQ helicases have similar recruitment kinetics to double strand breaks but significantly different retention kinetics. From these results, we can conclude that the mammalian RecQ helicases share some similarities but also play unique roles at sites of DNA damage. WRN is strongly associated with the nucleolus, as is nucleolin (NCL), an important nucleolar protein. Therefore, we have investigated if these nucleolar proteins interact and if this interaction has a functional significance. We showed that WRN does interact with NCL, and mapped the binding region to the C-terminal domains of both proteins. Furthermore, NCL, or its C-terminal fragment, inhibits the helicase, but not the exonuclease activity of WRN. These data suggest that NCL may regulate unwinding by WRN. We postulated that the NCL-WRN complex may contain an inactive form of WRN, which is released from the nucleolus upon DNA damage. Then, when required, WRN is released from inhibition and can participate in the DNA repair processes. Future studies aim to characterize more fully the protein complexes that WRN participates in both before and after DNA damage.
Showing the most recent 10 out of 55 publications
