1. Research Background:
Each living organism depends on the accurate maintenance of its genetic information. Genome maintenance mechanisms include DNA replication, which faithfuly copies the genetic information during S phase of each cell division, and DNA damage repair, which ensures genome integrity despite the onslaught of DNA damage from various sources. The process of DNA replication makes S phase particularly vulnerable to DNA damage. DNA damage and other replication impediments lead to the stalling of DNA replication – a phenomenon termed replication stress. Notably, the occurrence of replication stress is not only a hallmark of early cancer development, but replication stress is now understood as a key driver of cancer formation (1,2). Replication stress is, however, a broadly defined phenomenon with several distinct molecular causes. While sensitive markers of DNA damage exist, these do currently not allow to discriminate the molecular origins of replication stress thereby limiting the potential of therapeutics targeting genome maintenance mechanisms.
Single-stranded DNA (ssDNA) is formed during replication stress through several mechanisms. The location of this single-stranded DNA in the genome (the “ssDNA signature”) depends on the mechanism of ssDNA formation and the location of replication stall sites. In this project we aim to establish ssDNA signatures as marker of DNA replication stress and discriminator of its molecular causes. The method will then be used to characterize replication stress caused by different oncogenes as well as during the ageing process.
2. Research questions addressed by the group:
- 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?
- How are cellular DNA repair decisions made? Can DNA repair decisions be controlled from the outside in order to improve genome editing technology?
- 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):
Single-stranded DNA (ssDNA) forms during replication stress through several mechanisms including stalling of DNA polymerases, replication re-priming and resection of DNA breaks. We refer to the genomic profile of ssDNA as the ssDNA signature. This ssDNA signature depends on the molecular mechanism of ssDNA formation and the location of replication stall sites, which in turn depends on where in the genome replication had initiated and how much DNA synthesis has occurred. The ssDNA-signature is therefore a highly informative, quantitative read-out that will allow to reveal from which source replication stress has originated. Our previous work showed that ssDNA signatures can be obtained using chromatin-immunoprecipitation (ChIP) of the ssDNA-binding protein RPA, followed by preparation of strand-specific DNA libraries and next-generation sequencing (3). Furthermore, we recently showed in a proof-of-principle study that unscheduled DNA replication outside S phase induced replication stress with a characteristic ssDNA signature (4).
In the proposed project, we will use RPA-ChIP, but also develop new workflows to sequence ssDNA, which overcome current limitations of ChIP and allow us to determine boundaries of ssDNA fragments.
Using these methods, we will generate ssDNA signatures from budding yeast (as proof of principle) and human cells. Therefore, we will first (i) utilize genetic engineering and chemical disturbance to generate well-characterized scenarios of replication stress in both systems and identify the corresponding ssDNA-signatures. The relation of ssDNA-signatures to known chromosome features such as replication origins (yeast), transcription units and chromatin states will be mapped bioinformatically. Additionally, DNA synthesis will be measured by incorporation of the nucleotide analog EdU. Collectively, these experiments will reveal the relation of well-researched causes of replication stress and the resulting ssDNA signature. Second, (ii) we will then utilize ssDNA signatures to determine the origin of replication stress upon activation of selected oncogenes. Using the standards determined in (i) and in collaboration with local cancer biologists and oncologist, we will systematically investigate whether different oncogenes induce replication by a common or divergent mechanism. Third, (iii) we will use the yeast model to determine whether ageing cells suffer from replication stress and by which mechanism it arises. Budding yeast offers the advantage that a homogeneous popultation of aged organisms can be obtained using genetic systems (5). This offers us the possibility to investigate for the first time, whether the occurance of replication stress is not only a feature of cancer cells, but ageing organisms in general.
4. Applied Methods and model organisms:
- application and development of NGS workflows (Illumina, Oxford Nanopore), including development of new methods for enrichment of ssDNA and library preparation
- quantitative cell and molecular biology methods (microscopy (high-content), flow-cytometry, genome editing)
- computational analysis of NGS data
- human cell culture, 2D and 3D models of oncogenic transformation
- budding yeast (S. cerevisiae) as model for replicative ageing and for method development and proof-of-principle experiments
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 are 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 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 strictky required, as it can also be obtained in the course of the project
- Halazonetis TD, Gorgoulis VG, Bartek J (2008). An oncogene-induced DNA damage model for cancer development.
Science, 319(5868), 1352-1356.
- Kotsantis P, Petermann E, Boulton SJ (2018). Mechanisms of oncogene-induced replication stress: jigsaw falling into place.
Cancer discovery, 8(5), 537-555.
- 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
- 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)
- Lindstrom DL, Gottschling DE (2009). The mother enrichment program: a genetic
system for facile replicative life span analysis in Saccharomyces cerevisiae. Genetics, 183(2), 413-422.