figure research dominik boos

We work on three main questions in the lab:

  1. Upon DNA damage the DNA damage checkpoint inhibits firing of most origins to avoid mutations through copying damaged DNA templates. Counterintuitively, some origins that are normally dormant fire more efficiently upon DNA damage, showing that complex regulations must exist and need to be investigated. Our lab identifies and characterises regulators and regulating pathways of initiation by searching and characterising physical interactors and posttranslational modifications of the Treslin-TopBP1-MTBP complex.
    We identified the CDK8 kinase as an interactor of MTBP (Köhler et al. 2019). We showed that the interaction is required for complete genome duplication and for proper subsequent segregation of chromosomes in mitosis. We are now investigating in detail the cellular replication function and the molecular mechanisms of CDK8 that had not previously been implicated in DNA replication.

    A second project in the lab is to investigate MTBP phosphorylation. We found that MTBP is inhibited by phosphorylation. We are investigating the cellular roles of this inhibition and the molecular mechanisms involved.

    To investigate cellular replication we use various techniques in cultured human cells. Upon replacement of endogenous MTBP or Treslin with specific mutants using RNAi, we analyse replication by incorporation of nucleotide analogues and flow cytometry, fluorescence imaging or DNA combing. Replication stress is measured by quantifying sites of underreplication using classic cytogenetics or fluorescence microscopy. The impact of underreplication on subsequent chromosome segregation is investigated by microscopic quantification of normal and aberrant mitotic figures.

  2. Moreover, the lab will investigate the cellular and biochemical functions of the Treslin-MTBP-TopBP1 complex, and its regulation by the cell cycle kinases CDK and DDK, as well as by the DNA damage checkpoint (Fig.1). Cell cycle regulations are pivotal to facilitate the ‘temporal replication program’, the reproducible order of replication of the different chromosomal domains in every S phase. Checkpoint regulations ensure the proper management of replication under DNA damage to avoid changes of the genetic information.

  3. Motivated by the finding that cancer and Meier-Gorlin syndrome have been linked to lower levels/activity of replication initiation factors we are investigating the consequences of lowering the activity of MTBP and Treslin or other firing factors in cells using three approaches: 1) partial RNAi of MTBP/Treslin, 2) CRISPR/Cas9-mediated knock out of one MTBP/Treslin copy, 3) chemical inhibition of DDK. We observed that these treatments lead to replication stress already at low levels of origin firing inhibition. We intend to learn about the specifics of this replication stress by comparing it with classic replication stress induced by inhibitors of replication elongation, such as aphidicolin. Low-initiation induced replication stress could fundamentally differ from classic stress because: 1) lower amounts of replication forks may lead to lower replication stress checkpoint signalling, leaving the cell ill-prepared to deal with the stress, and 2) affecting MTBP, Treslin and other firing factors may differentially affect different subsets of origins in the genome, leading to specific consequences.