In summary, coding and non-coding TNRs in humans apparently share mechanisms of expansion. However, the cell type and its status of division impose constraints on which mechanisms are used. Shorter changes of premutation-length alleles can apparently occur in any cell type. Whether expansions, in some cases, are residual deletion products should be considered. However, longer expansions or deletions are observed under particular circumstances. In humans, the largest expansions of coding or non-coding alleles occur in quiescent cells and involve DNA repair-dependent mechanisms. Deletions of the largest alleles are most prominent in dividing cells. Thus, the biology of human disease not only provides insights into where and when repeat instability occurs but also suggests that expansion and deletion, of at least long alleles, occur by distinct mechanisms.
Many questions and puzzling features remain. However, to continue progress, there is a crucial need to draw conclusions from appropriate experimental systems that closely reflect the properties of the cell type in which expansion occurs. It will also be important to integrate the results of different experimental approaches, as they may lead to different conclusions. For example, the role of MMR in expansion is a work in progress that will benefit greatly by considering more than one set of data. The MSH2-G693A112
mouse model provides compelling evidence for the importance of ATPase activity in expansion. However, the biochemistry of the MSH2-G693A mutant protein needs to be explored further: does it bind ATP, does the mutation influence the activity of MSH3 or might the ATPase activity of MSH3 compensate for the mutation in MSH2? In vitro
repair assays are powerful systems for evaluating the enzymology of loop repair. However, cell extracts contain the machinery of many active DNA repair pathways, and discerning which ones are operating at the TNR loops is an important and challenging issue. Biochemical measurements of MMR and of other enzymes implicated in expansion need to be integrated with DNA repair assays to link enzymology with function.
In general, we are only beginning to think about the possibility of crosstalk among DNA repair pathways and their relationship with TNR expansion78
. However, tantalizing pieces of evidence suggest that hybrid pathways might be important. For example, MSH2–MSH6 and the BER glycosylase, MUTYH, form a physical complex117
. It can be speculated, though not yet demonstrated, that MSH2–MSH3 and OGG1 interact, which could explain the involvement of BER and MMR in TNR length changes. Future work that explores the interactions among the components of different repair pathways will be informative.
More attention needs to be paid to TLPs and their role in TNR instability. Interestingly, TLPs are involved in BER and NER — both of which are candidate pathways for expansion — and may also be involved in generating large deletions (in replication restart). In NER, the DNA damage binding proteins DDB1 and DDB2 (also known as XPE)118
, which recognize cyclobutane-pyrimidine-dimers (CPDs) and UV-induced (6-4) pyrimidine photoproducts, and Pol η and Q119,120
are TLPs. In BER, Pol λ serves as a back-up for OGG1 and has fivefold greater DNA synthesis fidelity than Pol β121,122
. Its use may result in robust flap formation if oxidized bases block Pol β progression at TNRs122
. The majority of evidence suggests that oxidized bases in random DNA sequences do not inhibit replicative DNA polymerases, but it will be important to test whether oxidized bases in CG-rich TNR tracts promote polymerase stalling. It should also be tested whether TLPs can promote strand displacement or enhance lengthening of a single-strand flap in either BER or TCR. The use of TLPs may be a common factor in large changes in TNR length and should be explored more thoroughly.
Finally, all of these mechanisms for expansion must operate within the context of chromatin, and there is growing interest in exploring how chromatin structure and epigenetic modifications influence expansion. For example, a study of the human ATXN7 locus in transgenic mice has established a link between binding of CCCTC-binding factor (CTCF, a regulatory protein implicated in DNA conformation and genomic imprinting) and regulation of repeat instability123
. Currently, the links among epigenetic changes and expansion remain enigmatic, but the influence of genome locus, post-translational modification of histones and DNA methylation on TNR expansion will be key issues to explore4,5,124,125
In conclusion, TNR expansions of ten to 10,000 units add a stunning 30 to 30,000 base pairs to DNA during transmission and during somatic growth, and both contribute to disease onset. Thus, blocking expansion at various developmental stages is likely to be beneficial. The inherited TNR tract determines whether an individual develops disease, but progressive somatic mutation may influence, at least in part, when disease occurs. The ability to modulate expansion raises hope that the severity of pathophysiology might be reduced or its onset delayed, thereby widening the therapeutic window for these deadly TNR diseases.