Repair of DNA double strand breaks by “alternative” DNA repair pathways
1 PhD project offered in the IPP summer call Molecular Mechanisms in Genome Stability & Gene Regulation
Scientific Background
The genomic integrity is extremely important for the normal cellular functions. However, cells are routinely challenged by various DNA damaging events leading to the formation of different DNA lesions. In addition to environmental (external) factors like UV, X-rays, ionizing radiations etc., cellular processes like hydrolysis, oxidation, alkylation, impediment to replication fork etc., can also damage DNA endogenously. Cells have evolved multiple DNA repair pathways to deal with these DNA lesions. DNA double strand breaks are one of the most complex and thus deleterious DNA lesions to repair. The major pathways used by cells to repair DSBs are non-homologous end-joining (NHEJ) and homologous recombination (HR). Usually, HR is considered “error-free” pathway as the broken DNA duplex finds and invades the sister chromatid (or homologous chromosome) to use it as template to copy and recover the broken DNA sequence. In comparison to HR, NHEJ is “error-prone” as it frequently requires processing (e.g., deletion) of broken ends before ligating them together. Importantly, the defective DSB repair can lead to cancer, immunological deficiencies, accelerated ageing and severe developmental abnormalities.
Besides HR and NHEJ, cells can also use other additional “alternative” DSB repair pathways in certain specific situations. Cancer cells experience DNA damaging stress due to faster replication rate and thus they often hijack alternative repair pathways to overcome such stress. Break-induced replication (BIR) is one of the homology-directed alternative DSB repair pathway, which itself is highly mutagenic. BIR usually occurs at the single-ended DSBs where the second end is missing to carry out the more “accurate” HR. The basic mechanism of BIR underlies other deleterious processes/events like MiDAS (mitotic DNA synthesis), microhomology-mediated BIR (MMBIR) and certain form of chromothripsis. BIR is also postulated to be responsible for generating gross chromosomal rearrangement (GCR) found in neurological and neurodevelopmental disorders known as Pelizaeus-Mezbacher disease (PMD) and MECP2 duplication syndrome respectively. Most importantly, a form of BIR, known as ALT (alternative lengthening of telomers), is used by cancer cells to extend and thus maintain the critical length of telomers to avoid cell death. Around 10-15% of all cancers use ALT for telomere extensions to achieve unrestricted proliferation of cancer cells.
PhD Project: Investigating break-induced replication repair by in vitro reconstitution system
Genetic instability (GIN), with high mutation rates and chromosomal rearrangement in pre-cancerous cells, enables these cells to achieve transformation and unrestricted growth. The error-prone repair of one-sided DSB via BIR is postulated to be one of the underlying causes of GIN. As explained above, BIR is highly mutagenic and exhibits ~2800-fold elevated mutation rates compared to normal DNA replication. This is largely due to its unusual mode of conservative replication of newly synthesized DNA, lack of the S-phase processive replisome and mismatch correction, and longer persistence of ssDNA. While yeast genetics have uncovered many features of BIR, its molecular mechanism and the details of mammalian BIR is poorly understood. The latter is especially difficult to study due to impressively large heterogeneity and stochastic nature of the BIR/ALT found in mammalian cells. To circumvent this issue, we aim to address this outstanding problem in our research group by reconstituting BIR in vitro for both yeast and mammalian system using bulk biochemistry and single-molecule imaging (SMI) techniques, supported by cell biology. This PhD project will involve characterizing the molecular mechanism of BIR in unprecedented details using purified proteins, ensemble biochemistry and SMI techniques such as optical dual tweezer system (C-trap, Lumicks) and TIRF (total internal reflection microscopy)-based platforms. Later, the in vitro findings will be supported and validated by cell biology-based assays. The establishment of BIR at single-molecule level will be transformative for the understanding of BIR mechanism and its role in cancer development
We are looking for motivated PhD student with strong interest in the mechanism of DNA damage response, genomic instability, and cancer development. Ideally, the interested candidate will have some prior experience of molecular biology for cloning, protein expression and purification, and biochemical assays. However, lack of experience in these techniques should not stop you from applying if you are strongly interested in learning the requisite skills. You bring your enthusiasm and determination; we will do the rest.
If you are interested in this project, please select Anand as your group preference in the IPP application platform.
Publications relevant to this project
Ranjha L, Howard S & Cejka P (2018) Main steps in DNA double-strand break repair: an introduction to homologous recombination and related processes. Chromosoma 127, 187–214 Link
Kockler ZW, Osia B, Lee R, Musmaker K, and Malkova A (2021) Repair of DNA Breaks by Break-Induced Replication. Annual Review of Biochemistry 90:1, 165-191 (2021) Link
Deem A, Keszthelyi A, Blackgrove T, Vayl A, Coffey B, et al. (2011) Break-Induced Replication Is Highly Inaccurate. PLOS Biology 9(2): e1000594 Link