Prof. Dr. Boris Pfander

Research Area: Genome Maintenance Mechanisms in Health and Disease

Branches: Cell BiologyGeneticsMolecular Biology

1. Research Background:

Each living organism depends on the accurate maintenance of its genetic information. Moreover, DNA damage and lack of genome maintenance are key drivers of ageing. DNA breaks (DNA double strand breaks (DSBs)) are a particularly severe form of DNA damage, as persistent DSBs strongly interfere with chromosome function and illegitimate DSB repair can lead to mutations and chromosome translocations. For these reasons DSB repair has to be efficient and accurate and eukaryotes have evolved several DSB repair mechanisms, each of which has the ability to fix the break. Therefore, at every DSB a cellular decision making process is triggered that determines how this particular DSB will be repaired (1). We know that the chromatin environment and the cell cycle state are crucial factors influencing this decision, but overall the decision making process called “DSB repair pathway choice” is poorly understood.

DSB repair is not only critical for cellular genome integrity, but also essential for all genome editing technologies (2). During genome editing CRISPR-Cas9 or related nucleases are used to induce DSBs at specific positions in the genome. Since repair of these DSBs entirely relies on endogenous DSB repair pathways, DSB repair pathway choice hereby determines the outcome of the genome editing reaction.

To study DSB repair pathway choice at the level of individual DSBs requires us to develop new methods and single-cell sequencing approaches in particular that allow to follow DSBs and their repair on a single-cell and single-molecule level. Such methods will allow us to study the DSB repair decision in diverse contexts, suchas at different chromosomal locations, during ageing (in models), after exposure to radiation (in patients and astronauts) and during genome editing.

2. Research questions addressed by the group:

  • How are cellular DSB repair decisions made? Can DSB repair decisions be controlled from the outside in order to improve genome editing technology?
  • What are the molecular causes of replication stress that drive cancer formation after oncogene activation? What is the contribution of replication stress to the ageing process?
  • What are markers of DNA damage that allow to quantify the exposure of human individuals to sources of DNA damage such as radiation?
  • How are genome maintenance mechanisms altered during ageing? How is their proteostasis controlled?

3. Possible project(s):

Eukaryotic organisms have an arsenal of repair mechanisms to mend DNA strand breaks (DSBs). How cells make the decision which of these repair mechanisms to use for an individual DSB is, however, poorly understood, despite a critical importance not only for genome stability, but also genome editing methodology. This is due to a lack of single cell methodology that allows to study the repair of individual DSBs.

A key step during DSB repair and DSB repair pathway choice is the formation of single-stranded DNA (ssDNA) in a process of DSB resection. The amount of ssDNA generated at an individual DSB is a good proxy for DSB repair pathway choice.

This PhD project centers around the development of methodology, single-cell methodology in particular to visualize and quantify the formation of single-stranded DNA by DSB resection. Our previous work showed that ssDNA at DSBs can be efficiently mapped using a workflow involving chromatin-immunoprecipitation (ChIP) of the ssDNA-binding protein RPA, followed by preparation of strand-specific DNA libraries andnext-generation sequencing (3). Furthermore, we showed that this method allows to characterize DNA breaks occurring during the process of DNA replication in cell ensembles (4).

In the proposed project, we will devise new strategies to purify and map single- stranded DNA in order to reach single-cell capability on different NGS platforms. Such a new experimental approach will required the side-by-side development of new analysis pipeline(s). To this end the candidate will work closely with local collaborators from the field of bioinformatics.

At first, we will test the method on enzyme-induced DSB systems, which generate site-specific DSBs at specific chromosomal locations and with high temporal synchrony and are therefore an ideal testing ground of the methodology. The single- cell approach will lay open new aspects that have previously been obscure, including the temporal dynamics. It will also reveal how the DSB repair decision is influenced by chromosome architecture and chromatin state.

Next we will apply this method on our own and with local and international collaboration partners to study how the DSB repair decision changes during (i) ageing and (ii) in response to radiation. For the ageingexperiments, we will use budding yeast models of replicative ageing (5), but also aim to test DSB repair in peripheral blood cells from human individuals. We will also investigate how repair is influenced by ionizing radiation, in particular heavy.ion radiation, which is highly relevant for radiation therapy as well as for the health of astronauts.

4. Applied Methods and model organisms:

Methods:

  • application and development of NGS workflows (Illumina, Oxford Nanopore), especially development of single-cell methods to measure DSBs and intermediates of DSB repair
  • quantitative cell and molecular biology methods (microscopy (high-content), flow-cytometry, genome editing)
  • computational analysis of NGS data

Models:

  • human cell culture
  • budding yeast (S. cerevisiae) as model for replicative ageing; to obtain proof-of- principle during method development
  • human primary blood cells

5. Desirable skills and qualifications:

We are looking for a biochemist or cell-biologist with a desire to reveal fundamental principles of biology and a strong interest in method development (NGS workflows). Prior experience in molecular biology and associated lab work is required.

A sound knowledge of fundamentals of molecular cell biology is required. Particular areas of interest should be genome maintenance, cancer development and ageing. The applicant should be able follow the scientific literature, to deduce and creatively apply the obtained knowledge on his/her own as well as in the context of a team and be able to efficiently communicate his/her obtained results in written and oral form.

A basic knowledge in programming (Python, R) will be useful, but is not strictly required, as it can also be obtained in the course of the project.

6. References:

  1. Symington, L.S. & Gautier, J. (2011). Double-Strand Break End Resection and Repair Pathway Choice.
    Annu. Rev. Genet. 45, 247-271.
  2. Gallagher, D.N. & Haber, J.E. (2018). Repair of a Site-Specific DNA Cleavage: Old-School Lessons for Cads9-Mediated Gene Editing.
    ACS Chem. Biol., 13, 397-405.
  3. Peritore M, Reusswig KU, Bantele S, Straub T, Pfander B* (2021). Strand-specific ChIP-seq at DNA breaks distinguishes single versus double-stranded DNA binding and refutes single-stranded nucleosomes.
    Mol Cell, 81(1), 1-13 doi: 10.1016/j.molcel.2021.02.005
  4. Reusswig KU, Bittmann J, Peritore M, Wierer M, Mann, M, Pfander B* (2021). Unscheduled DNA replication in G1 causes genome instability through head-to- tail replication fork collisions.
    Nat Commun, (in revision)
    bioRxiv https://doi.org/10.1101/2021.09.06.459115
  5. Lindstrom DL, Gottschling DE (2009). The mother enrichment program: a genetic system for facilereplicative life span analysis in Saccharomyces cerevisiae. Genetics, 183(2), 413-422.