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

Bakshi, K.; Nagamalleswari, E.; Breuker, J.; Eisenhardt, N.; Tiwari, R.; Gonzalez-Prieto, R.; Fatouros, C.; Chaugule, V.; Lee, H.; Ilic, D.; Trulsson, F.; Mittler, G.; Vertegaal, A. C. O.; Pichler, A.

doi:10.1101/2022.04.11.487769

bioRxiv · 10.1101/2022.04.11.487769

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

Bakshi, K.·Nagamalleswari, E.·Breuker, J.·Eisenhardt, N.·Tiwari, R.·Gonzalez-Prieto, R.·Fatouros, C.·Chaugule, V.·Lee, H.·Ilic, D.·Trulsson, F.·Mittler, G.·Vertegaal, A. C. O.·Pichler, A.

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Abstract

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.

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Bakshi, K., Nagamalleswari, E., Breuker, J., Eisenhardt, N., Tiwari, R., Gonzalez-Prieto, R., Fatouros, C., Chaugule, V., Lee, H., Ilic, D., Trulsson, F., Mittler, G., Vertegaal, A. C. O., Pichler, A.. 2022-04-11. Multi-level monitoring of EME1-MUS81 in CPT induced nucleolar rDNA repair. https://doi.org/10.1101/2022.04.11.487769

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