PhD Scientific Days 2022

Molecular Sciences I. (Poster discussion will take place in the Aula during the Coffee Break)

Genome-level analysis of DNA damage tolerance pathways in a human cell line

Text of the abstract

1. Introduction
Genetic information of living organisms is continuously challenged by intrinsic and extrinsic mutagenic agents like UV-radiation or reactive oxygen by-products of cellular metabolism. These agents change the chemical properties of DNA molecule, forming so-called genomic lesions. If replication machinery encounters an unrepaired lesion, the high-fidelity replicative polymerases are unable to recognise the changed genomic site and replication gets stalled. This is potentially dangerous state, as stalled replication forks tend to collapse, leading to a double strand break and genomic rearrangements. To avoid this, several DNA-damage (DDT) pathways have evolved, such as recombination-based template switch (TSw), translesion synthesis (TLS) which operates with low-fidelity translesion polymerases, repriming of replication and replication-fork protection.
2. Aims
Our aim was investigating molecular mechanisms of DTT pathways with mutant human cell line models utilizing modern genomic approaches.
3. Methods
We knocked out a set of DDT genes in RPE-1 P53 -/- cell line using CRISPR-Cas9 mutagenesis. Mutant clones were validated by Sanger-seq and (if it was possible) with western blot. Single and double mutant cell clones were cultured for 60 days then prior to the isolation of subclones. Genomes of three subclones and the ancestral clone were determined using next generation DNA sequencing. After bioinformatical processing of raw genomic data, we mined, analysed and compared mutagenic patterns found in different mutant cell clones.
4. Results
Our genomic and molecular results revealed that 1) There are two distinct TLS subpathways. The REV1-dependent TLS acts during replication, while REV1-independent TLS takes part in postreplicative gap filling. 2) In absence of homologous recombination (HR), replicative TLS is responsible for elevated mutagenesis. 3) REV1-dependent TLS and replication repriming are competitive pathways during replication. 4) Most of background mutagenesis in our cell model can be linked to TLS.
5. Conclusions
Our results highlight the importance of DDT pathways in the maintenance of the integrity of genome and show that replication-coupled DDT pathways are strongly interconnected to homologous recombination, indicating a new strategy for HR-deficient cancer therapy.
6. Funding
Cooperative Doctoral Program
Breast Cancer Research Fundation