Towards the identification of genetic factors influencing the stability of short tandem repeats

This work has identified three distinct approaches that can be used to identify genetic factors involved in the STR instability process. We propose that Schizosaccharomyces pombe, a yeast now recognised as being a good model for the study of many cellular processes in higher eukaryotes, is also suitable for the study of STR instability. We have demonstrated that in S. pombe a plasmid based (CAG/CTG)_n sequence containing between 25-74 repeat units is stable over approximately 55 mitotic divisions. A frame-dependent reporter gene construct, based on colour, has also been created containing the dinucleotide repeat (CA/GT)₁₂. By following the change in repeat length or colony colour (for the two constructs respectively) in strains containing both known or randomly created genomic disruptions/mutations it will be possible to identify factors that directly impact upon repeat length. One factor that we have considered as a potential candidate is the product of the S. cerivisiae postreplication repair gene RAD5. An increase in the stability of the dinucleotide repeat (CA/GT)_n has been demonstrated in strains deleted for this gene (Johnson et al., 1992). Discussed are the attempts that have been made to clone the S. pombe homologue in order to determine whether this function is maintained between species. Also identified, through the technique of Southwestern blotting, are a number of proteins that bind both single stranded [(CAG)₁₀, (CTG)₁₀, (CCG)₁₀, and (CGG)₁₀] and double stranded [(CAG/CTG)₁₀ and (CCG/CGG)₁₀] trinucleotide repeats. The interactions, with the (CCG)₁₀, oligonucleotide, of a group of proteins ranging in size from 18-45 kDa have been demonstrated to be specific. Further identification of these proteins and their function(s) in binding this particular sequence may identify a role in (CCG/CGG)_n instability and may in turn suggest other proteins or pathways to be investigated in the future. This work demonstrates that a number of methods are available for the study of STR instability. A combined approach based on the identification of i) novel sequences using reporter gene constructs; ii) proteins that interact with repetitive DNA and iii) candidate sequences, pathways and mechanisms elucidated in other organisms, will in the future, enable short tandem repeat instability to be more fully understood.