Typos in the genome: Defining the molecular mechanism of replication slippage

Given the broad role that replication plays in human health, defining how replisomes cope with challenging templates is of fundamental importance. While replication stress can be induced by chemicals or radiation, DNA itself has been implicated as an endogenous source of stress. Specifically, sequences that adopt various secondary structures⁵ (e.g., hairpins, cruciforms, G-quadruplexes (G4s), intercalated motifs (i-motifs), and triplexes) are hotspots for mutations and translocations across various cancers^6,7. However, such sequences play important biological roles and mechanisms must be in place to preserve them. A complete molecular understanding of how these sequences are preserved while maintaining genome stability is lacking. Furthermore, the causal relationship between perturbed replication dynamics, altered fidelity, and loss of genome integrity is unclear.

This proposal aligns well with the MRC DTP theme of “Genome Stability and DDR” and will impact a broad range of disciplines and fields, including nucleic acid chemistry and structure, cancer biology, genome evolution and repeat expansion disorders.

We have recently discovered that structure-forming sequences stall reconstituted budding yeast replisomes^8,9. This highlights the DNA template as a direct source of replication stress. To understand the relationship between altered replication dynamics and fidelity, we must first define the mechanisms that impact replication fidelity within repetitive sequences. This is the major goal of this proposed project.
 
Specific aims:
 
Determine the mechanism of replication slippage using reconstituted replisomes
 
Here, the PhD candidate will used a well-established approach in the lab which allows us to reconstitute DNA replication in a test tube using purified budding yeast proteins. This powerful approach generates mechanistic insight that cannot be obtained in other ways. A similar system has recently been developed for human replisomes, which will also be established in the lab.
To obtain nucleotide-resolution results, the PhD candidate will employ denaturing sequencing gels. Comparing the length and distribution of replicated products from different templates and with different reaction conditions will allow us to determine fundamental aspects of replication slippage.
 
Directly sequence in vitro replicated products using single molecule PacBio sequencing
 
We have an onoging collaboration with the lab of Prof. Vincent Dion (University of Cardiff). The Dion lab have developed an experimental and bioinformatic pipeline for single-molecule sequencing of repetitive sequences¹⁰. Identifying rare mutations within repeats using conventional sequencing is very difficult as repeat are often filtered away or generate too much noise. However, these challenged can be resolved using single-molecule sequencing, such as that obtained by the Pacific Biosciences (PacBio) platform. The Dion lab have an in-house Sequel IIe platform and have developed an algorithm to identify mutations within complex repetitive sequences.
 
Here, the PhD candidate will collaborate with the Dion lab to directly sequence in-vitro replication products. We have devised an approach to specifically sequence replicated material whilst also differentiating leading versus lagging strand products. This will provide direct evidence of replication slippage and will also pinpoint exactly which mutations are produced, where and at what frequency.
 
Establish how local DNA context affects replication slippage
 
There is evidence that the local DNA sequence around a repetitive sequence can affect the mutational outcomes of replication slippage¹¹. Why or how this is the case is unclear.
To determine the effect of local DNA context, the PhD candidate will clone and test the effect of different flanking 5’ and 3’ sequences derived from the human genome and whether this is modulated by the replicative polymerase used.
 
Develop a high-throughput single-molecule approach to study replication fidelity in vitro
 
Since there is limited scope in studying a small subset of repeats and sequence contexts, an ideal approach would be a high-throughput assay where a library of repeats is replicated and then sequenced using single-molecule PacBio platforms. Thus, the PhD candidate will develop a method for replicating and sequencing a library of barcoded repeats with a large variety of flanking sequence contexts. This will provide a broad understanding of the sequence determinants that drive replication slippage.
 
Altogether, this project will provide a mechanism for replication slippage while establishing state-of-the-art biochemical approaches. It will identify which replisome components and DNA sequences play a role. The results are relevant not only for all MSI cancers but also beyond, impacting the fields of genome stability and cancer evolution.

B.Sc. or M.Sc. (First or 2:1, or equivalent) in any of the following disciplines:

- Life Sciences / Biology

- Biochemistry

- Biomedical Sciences

- Molecular Biology

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