Research

Our lab aims to understand the regulatory mechanisms which control DNA replication during a normal cell cycle and in the presence of DNA damage. DNA replication is the principle process, which in every living organism allows the genetic information to be copied accurately and inherited to the next generation. This is a challenging task, which requires a complex regulatory network in order to ensure that DNA is duplicated fast, accurately, to completion, once per cell cycle and coordinated with other cellular processes that are essential for inheritance of genetic and epigenetic information. Failure of this regulatory network leads to genomic instability - the cause of cancer.
We use the yeast Saccharomyces cerevisiae as a model system, which offers the advantage of using elegant genetic tools in combination with quantitative biochemical methods and modern genomic and proteomic approaches.

The molecular machines that catalyze DNA replication are called replisomes. Replisomes are multi-protein complexes, which are specifically assembled at hundreds of DNA elements called origins of replication. Various essential catalytic activities are present in replisomes such as the Mcm2-7 DNA helicase or DNA polymerases. Other components have no known catalytic activity. We propose that a major function of these proteins is to facilitate regulation and coordination with other pathways such as the DNA damage response, chromatin assembly or chromatid cohesion. We investigate protein-protein interactions of these replication proteins in order to better understand replisome organization. In particular we focus on post-translational modifications, which are key to the regulation of DNA replication.

Every stretch of DNA has to be replicated precisely once per cell cycle and accordingly each replication origin is regulated to fire once only. Replication initiation is therefore separated into two steps: Mcm2-7 - the replicative DNA helicase - is loaded at origins in an inactive form during G1 and activated in S-phase. Two kinase complexes - CDK and DDK (Cdc7-Dbf4) - become active at the G1/S boundary and are required for helicase activation. Since CDK inhibits helicase loading at the same, these kinases ensure the principle “single-shot” regulation of DNA replication. We know the minimal set of phosphorylation substrates that is required for helicase activation (CDK: Sld2, Sld3; DDK: Mcm2-7), but how activation is achieved is not understood at a mechanistic level. Another regulatory feature of DNA replication remains completely enigmatic: activation of different origins does not occur simultaneously, but follows a temporal order. We aspire a mechanistic understanding of these different levels of regulation.

A particular focus of our research is the Dpb11 protein, which facilitates the CDK regulation of replication initiation. Dpb11 contains two pairs of phospho-protein binding BRCT domains, via which it interacts with CDK-phosphorylated Sld2 and Sld3. Mechanistically Dpb11 is thought to bridge the Sld2- and Sld3-subcomplexes and in this way lead to the formation of a replisome and activate the helicase. Our aim is to understand this crucial regulation and potentially characterize additional components.

The DNA replication process makes the cell vulnerable to the presence of DNA damage, since each single DNA strand is required to contain the genetic information in an undamaged form that can be read by DNA polymerases. Furthermore, if replication is stalled at a DNA lesion over a prolonged period of time, replication forks may degrade to potentially deleterious double-strand breaks. Therefore, DNA replication and cell cycle progression are controlled by checkpoint mechanisms. These signal transduction pathways can directly sense the presence of DNA lesions or replication problems and lead to the stabilization of stalled forks, the delay of activation of further replication origins and the arrest of the cell cycle. Interestingly, Dpb11 has a second role as mediator of checkpoint signaling. Similar to its role in DNA replication initiation it forms a ternary complex with the checkpoint proteins Rad9, the 9-1-1 complex and Mec1-Ddc2. Our experiments aim to understand checkpoint signaling mechanistically and investigate potential links between the two functions of Dpb11 in replication initiation and the checkpoint – two processes that are known to be regulated interdependently.