The human genome is littered with repetitive, low complexity sequences. Variation in the length of these sequences comprise one of the most potent sources of human genetic diversity and impacts gene expression and phenotypic diversity. However, the mechanisms that regulate repeat length are poorly understood. Repetitive sequences pose a problem for DNA replication as they can cause polymerase slippage, in which the DNA polymerase loses register in the repeat, leading to gain or loss of units of the repeat. In addition, some repeat sequences are also capable of forming non-B-form secondary structures, which act as impediments to DNA synthesis, which may also be associated with mutagenesis [1, 2].
A key focus of the lab has been to understand why some DNA sequences become frequent structural impediments to replication and to characterise the mechanisms that allow cells to detect and process such structures during replication [see 3 & 4 for recent examples]. We have proposed that the regulation of repeat length is dependent upon a balance between replication slippage and mutagenesis associated with structure-induced replication stalling, which are in turn a function of the nature of the repeat sequence [5]. However, some sequences do not conform to this general model and occasionally escape this homeostatic mechanism to expand enormously in a relatively small number of somatic cell divisions. Such repeat expansions have been linked to human neurodegenerative diseases but the mechanisms that drive these aberrant expansion events remain unknown.
The project will use genome editing in human stem cells and iPSCs from patients, coupled with cutting edge sequencing and structural genomic methods, to monitor repeat stability during differentiation in controlled genetic backgrounds to explore the mechanisms that result in the dramatic expansion of certain repeat sequences involved in diseases such as Amyotrophic Lateral Sclerosis and Friedreich Ataxia.
References
- Lerner, L.K. and Sale, J.E. (2019).
Replication of G quadruplex DNA.
Genes (Basel) 10(2). pii E95. doi: 10.3390/genes10020095 - Mellor, C., Perez, C. & Sale, J.E. (2022).
Creation and resolution of DNA structural impediments during DNA replication.
Critical Reviews in Biochemistry & Molecular Biology in press. - Šviković, S., Crisp, A., Tan-Wong, S.M., Guilliam, T.A., Doherty, A.J., Proudfoot, N.J., Guilbaud, G. and Sale J.E. (2019).
R-loop formation during S phase is restricted by PrimPol-mediated repriming.
EMBO J. 38(3). pii: e99793, doi:10.15252/embj.201899793. - Lerner, L.K., Holzer, S., Kilkenny, M.L., Murat, P., Šviković, S., Schiavone, D., Bittleston, A. Maman, J.D., Branzei, D., Stott, K., Pellegrini, L. and Sale, J.E. (2020).
Timeless couples G quadruplex detection with processing by DDX11 helicase during DNA replication.
EMBO Journal 39, e104185. doi: 10.15252/embj.2019104185. - Murat, P., Guilbaud, G. and Sale, J.E. (2020).
DNA polymerase stalling at structured DNA constrains the expansion of Short Tandem Repeats.
Genome Biology 21, 209. doi: 10.1186/s13059-020-02124-x.