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Cell cycle regulation of structure-selective endonucleases during homologous recombination
Cell cycle regulation of structure-selective endonucleases during homologous recombination
The eukaryotic cell cycle is a complex process that coordinates protein function with the changing requirements of the different cell cycle phases. Many proteins are therefore regulated in a cell cycle-specific manner to make them available/active at a specific cell cycle phase, or prevent their action at other phases. Two proteins regulated in such a cell cycle-specific manner are the structure-selective endonucleases (SSEs) Mus81-Mms4 and Yen1 – repair factors required for the removal of DNA structures arising during homologous recombination (HR). Research in the last years thereby identified a variety of regulatory pathways leading to cell cycle-specific upregulation of the Mus81-Mms4 and Yen1 catalytic activity during M-phase. Despite accumulating evidence that the catalytic activity of the two SSEs is cell cycle-regulated, it remained elusive at which cell cycle phase they would exhibit their key function and how the different regulatory mechanisms upregulating Mus81-Mms4 and Yen1 during M-phase are working together. To address these questions, we developed an advanced toolbox of cell cycle tags which allowed us to restrict the expression of Saccharomyces cerevisiae Mus81-Mms4 and Yen1 to different cell cycle phases and thus analyze at which cell cycle phase these SSEs exhibit their key function. The advanced toolbox of cell cycle tags generally refines the methodology of cell cycle tags and overcomes critical limitations observed for previous cell cycle tag systems, such as the limited number of cell cycle tag constructs that did not allow adaption of expression levels. We circumvented this problem using genetic approaches like chimeric protein fusions, 5´UTR truncations and out-of-frame ATGs which resulted in a toolbox of 46 cell cycle tag constructs with a broad range of expression levels. In general, these advancements will help to answer the question of cell cycle regulation for many proteins and, more specifically, allowed us to address this question for the SSEs Mus81-Mms4 and Yen1. Applying the advanced cell cycle tag toolbox to Mus81-Mms4 and Yen1, we were able to restrict their expression to different cell cycle phases and attribute their key function to M-phase. Furthermore, we used the approach to reinstall cell cycle restriction to deregulated SSE versions, which highlights the importance of restricting SSE function to M-phase as their premature function during S-phase interferes with replication progression. As such, the observed function in M-phase matches the temporal regulation of the catalytic activity of Mus81-Mms4 and Yen1 which has been shown to be high in M-phase. For Mus81-Mms4, this upregulation of the catalytic activity is known to depend on phosphorylation by the cell cycle kinases CDK (cyclin-dependent kinase) and Cdc5 as well as on the formation of a scaffold protein complex. Here, we add a new kinase – the cell cycle kinase DDK (Dbf4-dependent kinase) – to this cell cycle regulatory network and gain insights into the interplay between the regulatory mechanisms involved. We establish that the two regulatory pathways, phosphorylation and scaffold protein complex formation, are highly interdependent and imply a switch-like activation mechanism. Taken together, our studies contribute to the understanding of the cell cycle regulation of Mus81-Mms4 and Yen1 and introduce an advanced toolbox of cell cycle tags which provides a technical source for studying cell cycle-regulated processes in general.
DNA repair, homologous recombination, cell cycle tags
Bittmann, Julia
2021
English
Universitätsbibliothek der Ludwig-Maximilians-Universität München
Bittmann, Julia (2021): Cell cycle regulation of structure-selective endonucleases during homologous recombination. Dissertation, LMU München: Faculty of Biology
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Abstract

The eukaryotic cell cycle is a complex process that coordinates protein function with the changing requirements of the different cell cycle phases. Many proteins are therefore regulated in a cell cycle-specific manner to make them available/active at a specific cell cycle phase, or prevent their action at other phases. Two proteins regulated in such a cell cycle-specific manner are the structure-selective endonucleases (SSEs) Mus81-Mms4 and Yen1 – repair factors required for the removal of DNA structures arising during homologous recombination (HR). Research in the last years thereby identified a variety of regulatory pathways leading to cell cycle-specific upregulation of the Mus81-Mms4 and Yen1 catalytic activity during M-phase. Despite accumulating evidence that the catalytic activity of the two SSEs is cell cycle-regulated, it remained elusive at which cell cycle phase they would exhibit their key function and how the different regulatory mechanisms upregulating Mus81-Mms4 and Yen1 during M-phase are working together. To address these questions, we developed an advanced toolbox of cell cycle tags which allowed us to restrict the expression of Saccharomyces cerevisiae Mus81-Mms4 and Yen1 to different cell cycle phases and thus analyze at which cell cycle phase these SSEs exhibit their key function. The advanced toolbox of cell cycle tags generally refines the methodology of cell cycle tags and overcomes critical limitations observed for previous cell cycle tag systems, such as the limited number of cell cycle tag constructs that did not allow adaption of expression levels. We circumvented this problem using genetic approaches like chimeric protein fusions, 5´UTR truncations and out-of-frame ATGs which resulted in a toolbox of 46 cell cycle tag constructs with a broad range of expression levels. In general, these advancements will help to answer the question of cell cycle regulation for many proteins and, more specifically, allowed us to address this question for the SSEs Mus81-Mms4 and Yen1. Applying the advanced cell cycle tag toolbox to Mus81-Mms4 and Yen1, we were able to restrict their expression to different cell cycle phases and attribute their key function to M-phase. Furthermore, we used the approach to reinstall cell cycle restriction to deregulated SSE versions, which highlights the importance of restricting SSE function to M-phase as their premature function during S-phase interferes with replication progression. As such, the observed function in M-phase matches the temporal regulation of the catalytic activity of Mus81-Mms4 and Yen1 which has been shown to be high in M-phase. For Mus81-Mms4, this upregulation of the catalytic activity is known to depend on phosphorylation by the cell cycle kinases CDK (cyclin-dependent kinase) and Cdc5 as well as on the formation of a scaffold protein complex. Here, we add a new kinase – the cell cycle kinase DDK (Dbf4-dependent kinase) – to this cell cycle regulatory network and gain insights into the interplay between the regulatory mechanisms involved. We establish that the two regulatory pathways, phosphorylation and scaffold protein complex formation, are highly interdependent and imply a switch-like activation mechanism. Taken together, our studies contribute to the understanding of the cell cycle regulation of Mus81-Mms4 and Yen1 and introduce an advanced toolbox of cell cycle tags which provides a technical source for studying cell cycle-regulated processes in general.