DNA replication is a process essential to every living organism. Replication stress can lead to double strand breaks and replication fork collapse, ultimately promoting cancer. The (CAGAGG)n DNA repeat (where n is 80-100) has been identified in a bioinformatics study of the mouse genome as heavily associated with DNA breaks. We hypothesize that this repeat forms a stable secondary structure, capable of blocking DNA replication enzymes. Based on our earlier biophysical data we proposed that (CAGAGG)n repeat forms a tetrastranded, antiparallel structure with two G×C×G×C tetrads stacked on top of each other and connected by A×G×A×G loops. In order to test this model and gain further structural details, we have designed 24 N-to-T single point mutants of the shortest sequence capable of forming stable secondary structure, 5’-AGG(CAGAGG)3CAG-3’ (also termed 1Core). The folding and thermal stability of mutants were analyzed via Circular Dichroism (CD) and UV-vis spectroscopies, and PAGE. Our results show that mutating any nucleotide in the G×C×G×C core destabilizes the structure by &gt; 15°C and is detrimental to its stability. Mutating A×G×A×G loops changes the stability of the structure only mildly, by no more than 5 °C. Our mutagenesis study strongly supports the model described above. In order to understand how 1Core structural units combine into a longer, more biologically relevant higher order structure, we have studied a 2Core sequence, 5’-AGG(CAGAGG)7CAG-3’. We proposed two possible arrangements of 1Core units into 2Core structure. We complement our extensive CD and UV-vis studies of 2Core with the use of 2 amino-purine (2AP) fluorescence to differentiate between the two models The environment of some adenines occupying the same position in the 2Core sequence, differs significantly in the contending models. We make use of the ability of 2AP to fluoresce while exposed to solvent; the florescence is quenched, however, in crowded environments (such as when 2AP is stacked). Combined our data shed light on the secondary structure of (CAGAGG)n repeat and, more generally, have great utility for understanding the secondary structures of intrinsically difficult-to-replicate repeat sequences. Such information can lead to the development of future anticancer therapeutics.

Pursuing the secondary structure of cancer-related DNA repeats using biophysical methods

256th National Meeting (2018)

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