Contact

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Dr. Boris Pfander
Group Leader
Phone:+49 89 8578-3050

MPI of Biochemistry, Am Klopferspitz 18, 82152 Martinsried

www.biochem.mpg.de/pfander

DNA Replication & Genome Integrity

Research

I – DNA Replication Control

Our lab aims to understand the regulatory mechanisms, which control DNA replication, the principal process, which in every living organism allows an accurate copying and inheritance of the genetic information to the next generation. This requires a complex regulatory network to ensure that DNA is duplicated fast, accurately, to completion, once per cell cycle and coordinated with other cellular processes. 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 and contain several essential catalytic activities such as the Mcm2-7 DNA helicase or DNA polymerases. DNA Replication control therefore operates by regulating the assembly and activity of replisomes.

Every stretch of DNA has to be replicated precisely once per cell cycle and accordingly each replication origin is regulated to fire only once.  Replication initiation is therefore separated into two steps: licensing and firing. During licensing, the Mcm2-7 helicase is loaded at origins as inactive precursor. During firing, these helicase precursors are activated by the association of accessory helicase subunits leading to the formation of active replisomes. Notably, firing and licensing are under control of two cell cycle kinases (CDK and DDK) and are thereby coupled to distinct cell cycle phases. This mutual exclusivity is the basis of the ‘single-shot’ regulation of DNA replication.

When investigating the temporal order of these phosphorylation events, we have observed that licensing and firing phases are separated by temporal gaps at cell cycle transitions and have characterized mechanisms that contribute to it. Importantly, experimental perturbation of the length of these gaps leads to the formation of chromosomal rearrangements, originating from sporadic over-replication events. These findings therefore emphasize that eukaryotic cells critically rely on stringent replication control in order to achieve once-per-cell-cycle-replication and genome integrity.

II – Control of Genome Integrity

Genome integrity is furthermore threatened by frequent DNA damage from various sources. Repair has to occur promptly and coordinated with cell cycle progression to avoid aggravation of the problem, but chromosomes undergo large structural changes during the cell cycle. We strive to obtain a general picture of how cells control the DNA damage response and adjust it to the cell cycle phase-specific features of chromosomes.

Repair of double-strand breaks by homologous recombination (HR) is under particular stringent cell cycle control, since (A) HR critically depends on the presence of a sister chromatid, (B) recombination intermediates interfere with chromatid segregation during M phase, and (C) nucleases involved in HR may interfere with S phase. Different steps of the HR reaction are hence cell cycle-controlled, but not all targets and regulators are known.

For HR initiation by DNA end resection in DFG-funded work we have uncovered a cell cycle-dependent regulatory pathway, which impinges on the nucleosome remodeller Fun30. Our data shows that transformation of damaged chromatin into a resection-permissive state by Fun30 functions as a critical bottleneck in the cell cycle control of resection. Notably, as we were able to uncouple Fun30 from its cell cycle control, we have made a step towards uncoupling DNA end resection from its cell cycle control, which will likely have important consequences also for the application of HR-based methodology, for example in CRISPR-Cas9-based gene editing. Furthermore, in the context of the CRC1064, we will now reveal the attributes and mechanisms underlying resection-permissive and resection-restrictive chromatin using systems biology and biochemical approaches.

For the last step of HR, we have identified new factors that regulate resolution of repair intermediates by the Mus81-Mms4 nuclease, which lead to a cell cycle-specific activation of Mus81 in M phase. These factors are on the one hand an M phase-specific cell cycle kinase complex (DDK-Cdc5) and an M phase-specific multi-protein complex involving several scaffold proteins. Overall, the activation of the Mus81-Mms4 bears many features of a cell cycle switch and we are currently investigating the significance of this control.

 
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