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Eisenhardt, N.

Publications and source records attributed to Eisenhardt, N..

2 recordsLinked to original sources

UBC9 and EME1 sumoylation foster ribosomal DNA damage repair in CPT response

UBC9 sumoylation (S*UBC9) in mammals contributes to SUMO target discrimination, biochemically1. Here, we present biological insights by characterizing a sumoylation mimetic UBC9-fusion (mCS~UBC9) in comparison to its wild-type (mCUBC9). We observe that sumoylation promotes UBC9s stability, nuclear localization and is beneficial for cell survival. We identified EME1, the regulatory subunit of the structure-specific endonuclease EME1-MUS81, as S*UBC9 substrate and demonstrate EME1 and UBC9 sumoylation being advantageous for survival upon Camptothecin (CPT) exposure. Moreover, mCS~UBC9 expression enhances double-strand breaks (DSBs) and replication upon CPT treatment, features assigned to the EME1-MUS81 complex2. EME1 and mCS~UBC9 co-localize in nucleolar repair-condensates, that enlarge and round when exposed to the drug. Together, these findings imply that S*UBC9-induced EME1 sumoylation improves ribosomal rDNA repair, which might prevent dangerous second template switches in highly repetitive ribosomal chromatin and allow the converging replication fork to complete DNA replication. Finally, we discuss implications of these findings for anti-cancer therapy.

molecular biology↗

Multi-level monitoring of EME1-MUS81 in CPT induced nucleolar rDNA repair

We identified EME1, the regulatory subunit of the structure-specific endonuclease complex EME1-MUS81, as substrate for the sumoylated UBC9 and demonstrated synergistic functions in promoting Camptothecin (CPT)-induced nucleolar ribosomal DNA (rDNA) repair (Nagamalleswari et al, co-submitted). Sumoylation of EME1 appears complex involving mono- and poly-sumoylation. Hence, we addressed here whether these modifications differentially regulate EME1 functions by analyzing EME1-variant expressing cell lines including mono-sumo(1)ylation and poly-sumo(2)ylation mimetic fusions. We complemented our analysis with the regulated endogenous EME1 interactome and our observation that Trichostatin A (TSA) induced EME1 and UBC9 sumoylation. Our findings are substantiated by identifying several regulatory proteins and by detecting CPT-induced endogenous di- and poly-sumoylated EME1 in different cell fractions. Together, our data suggest that Histone H4 acetylation, two mono-sumoylation events, poly-sumoylation, ISG15 and ubiquitination sequentially and in mutual dependence tightly control the intracellular localization, recruitment to DNA lesions, enzymatic activity, withdrawal from DNA lesions and the stability of EME1.

molecular biology↗