%0 Generic
%A Bohlmann, Mark
%D 2011
%F heidok:12824
%K SMUDO , SMCHD1 , DNA-damage response , HIPK2 , SMC proteins
%R 10.11588/heidok.00012824
%T Characterization of the SMC-hinge and MutL-homology domain (SMUDO) protein
%U https://archiv.ub.uni-heidelberg.de/volltextserver/12824/
%X The recently discovered SMC-hinge and MutL-homology domain (SMUDO) protein was found to be a potential interaction partner of the stress induced homeodomain-interacting protein kinase 2 (HIPK2) which acts as a switching element in the DNA damage response. Upon severe DNA damage, HIPK2 induces apoptosis via p53-dependent as well as p53-independent pathways. SMUDO protein exhibits structural domains showing homology with the SMC and MutL protein families, both known for their involvement in DNA repair mechanisms.  The aim of this thesis was to characterize the SMUDO protein and to investigate its putative connection to HIPK2 as well as its possible functions in the context of the DNA damage response.  Early analysis by Northern blot, RT-PCR and Western blot revealed ubiquitous expression of SMUDO in human tissue samples as well as in diverse cell lines, suggesting functions that are not restricted to specific tissues. Various DNA damage pathways have been studied to find indications for a role of SMUDO, where the focus was on those in which HIPK2 participates. Several kinds of DNA damage, such as double or single strand breaks as well as pyrimidine dimers, were generated by Adriamycin treatment or UV, stimuli which are known to activate HIPK2. However, SMUDO levels showed no alterations under these conditions, and SMUDO knockdown had no effect on levels of HIPK2, p53 phosphorylated at Ser46 or other DNA damage response transducers.  Pulldown experiments concerning HIPK2-SMUDO interaction support in vitro binding of the proteins, whereas in vivo interaction could not be confirmed by co-immunoprecipitation or immunofluorescence so far. This is probably due to SMUDO association to an insoluble fraction inside the nucleus, presumably chromatin. By FRAP technique, it could be shown that the SMC-hinge domain and the MutL domain of SMUDO influence the localization and mobility of the protein. The MutL domain seems to be responsible for a primarily nuclear localization of the protein and association with the insoluble nuclear fraction, while the SMC-hinge domain supports this association further limiting the mobility of SMUDO within the nucleus. Moreover, it could be demonstrated by co-immunoprecipitation that the SMC-hinge domain mediates SMUDO-SMUDO interaction.  Summarized, a SMUDO-HIPK2 interaction in the context of the DNA damage response seems to be unlikely. However, functional cooperation of the proteins might take place within other cellular processes such as epigenetic regulation.