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. It will also unwind unusual DNA structures such as triple helices and DNA forks. We are comparing the Werner helicase activity to that of another helicases in the family of RecQ helicases that are all involved in the maintenance of genome stability. WRN has another enzymatic activity, a 3-5'exonuclease function. We are searching for pathways in which WRN participates and have discovered a number of new functional and physical protein interactions with Werner protein. Our data strongly suggest that WRN is involved in two of the major DNA repair pathways: base excision repair and recombination. This conclusion is supported by biochemical studies of protein functional interactions and by cell biological data. Further, our observations and results from others suggest that a major function of WRN is at the telomere ends. WRN interacts with key telomeric proteins such as TRF1 and 2 and POT1. Since telomeres are G-rich, they accumulate 8-oxodG after oxidative stress and during the normal aging process. To investigate the role that WRN, BLM, and RecQ5 might play in telomere maintenance when telomeric DNA is damaged, the catalytic activities of these RecQ helicases were evaluated on 8-oxodG-modified telomeric D-loop substrates. Our results revealed that WRN and BLM preferentially unwound 8-oxodG-modified telomeric D-loops. Additionally, POT1 DNA binding was assessed in these studies and we showed that POT1 bound with higher affinity to 8-oxodG-modified telomeric D-loops but shows no preference for 8-oxodG-modified telomeric single-stranded DNA. Additionally, we analyzed the in vitro and in vivo interactions between WRN and DNA PKcs. DNA PKcs is well known to function in double strand break repair. Normally telomeres are not recognized as double strand breaks in cells however when telemeres become critically short telomere ends are recognized and this leads to the activation of DNA PKcs and a double strand break repair response. Our results showed that DNA-PKcs selectively stimulated WRN helicase but not WRN exonuclease activity in vitro on a model telomeric D-loop model. Additionally, we showed that overexpression of WRN could reverse the erosion of the telomeric G-tails normally found in DNA-PKcs knockdown cells. This study was the first to show that WRN and DNA PKcs cooperate to maintain normal telomeric DNA length. The functional interaction between DNA PKcs and WRN may have important implications for normal aging, because cellular senescence is also associated with shorted telomeric G-tails. Together, these observations extend our previous analysis of WRNs function at the telomeres and suggest that WRN protein is involved in the maintenance of damaged and undamaged telomere ends. WRN is also involved in the DNA repair processes. Specifically, we find in vitro and in vivo evidence for a role of WRN in the DNA repair of oxidative DNA base lesions. Our recent work has focused on the interaction of WRN with NEIL1. NEIL1 is response for the removal of several oxidatively generated DNA lesions, most notable the formamidopyrimidines. Others have shown that inactivation of NEIL1 leads to gastric cancers which strongly suggest that the activities of NEIL1 are critical for repair of formamidopyrimidines and other ring opened base lesions. We previously reported that NEIL1 and WRN interacted and that WRN stimulated NEIL1 incision activity. This study was extended to the other four RecQ helicases and we show that the NEIL1:WRN interaction is specific as no other RecQ helicase can similarly stimulate NEIL1. We further show that this stimulation requires a double-stranded DNA substrate and this finding may be important because NEIL1 can also work on lesions imbedded in single stranded DNA substrates. This study was one of the first to compare and contrast all five human helicases in one study and through such analysis we seek to gain a better understanding of which cellular functions are unique or shared among the RecQ helicases.

Agency
National Institute of Health (NIH)
Institute
National Institute on Aging (NIA)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIAAG000721-02
Application #
8156781
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
2
Fiscal Year
2010
Total Cost
$594,167
Indirect Cost
Name
National Institute on Aging
Department
Type
DUNS #
City
State
Country
Zip Code
Maity, Jyotirindra; Das, Biswadip; Bohr, Vilhelm A et al. (2018) Acidic domain of WRNp is critical for autophagy and up-regulates age associated proteins. DNA Repair (Amst) 68:1-11
Lee, Mina; Shin, Soochul; Uhm, Heesoo et al. (2018) Multiple RPAs make WRN syndrome protein a superhelicase. Nucleic Acids Res 46:4689-4698
Shamanna, Raghavendra A; Croteau, Deborah L; Lee, Jong-Hyuk et al. (2017) Recent Advances in Understanding Werner Syndrome. F1000Res 6:1779
Shamanna, Raghavendra A; Lu, Huiming; Croteau, Deborah L et al. (2016) Camptothecin targets WRN protein: mechanism and relevance in clinical breast cancer. Oncotarget 7:13269-84
Khadka, Prabhat; Croteau, Deborah L; Bohr, Vilhelm A (2016) RECQL5 has unique strand annealing properties relative to the other human RecQ helicase proteins. DNA Repair (Amst) 37:53-66
Shin, Soochul; Lee, Jinwoo; Yoo, Sangwoon et al. (2016) Active Control of Repetitive Structural Transitions between Replication Forks and Holliday Junctions by Werner Syndrome Helicase. Structure 24:1292-1300
Tippana, Ramreddy; Hwang, Helen; Opresko, Patricia L et al. (2016) Single-molecule imaging reveals a common mechanism shared by G-quadruplex-resolving helicases. Proc Natl Acad Sci U S A 113:8448-53
Khadka, Prabhat; Hsu, Joseph K; Veith, Sebastian et al. (2015) Differential and Concordant Roles for Poly(ADP-Ribose) Polymerase 1 and Poly(ADP-Ribose) in Regulating WRN and RECQL5 Activities. Mol Cell Biol 35:3974-89
Edwards, Deanna N; Machwe, Amrita; Chen, Li et al. (2015) The DNA structure and sequence preferences of WRN underlie its function in telomeric recombination events. Nat Commun 6:8331
Czerwi?ska, Jolanta; Pozna?ski, Jaros?aw; D?bski, Janusz et al. (2014) Catalytic activities of Werner protein are affected by adduction with 4-hydroxy-2-nonenal. Nucleic Acids Res 42:11119-35

Showing the most recent 10 out of 55 publications