Studying DNA Polymerase Slippage at Simple Sequence Repeats

Abstract

Short tandem repeats (STR) or simple sequence repeats (SSR), or microsatellites are 1-6 nucleotide or sometimes 1-10 nucleotide long repetitive motifs of DNA. These regions of DNA are known to be prone to de novo mutations, occurring during replication. Encountering SSR during replication, DNA polymerases slip or stall, hypothesised to be because of formation of secondary structures by the single stranded DNA. During replication, at SSR, polymerases transiently detaches, and with it, the newly formed strand, from the parent strand at the SSR and anneals again and polymerase starts replication further. During this process, often the SSR lead to mismatch during re- annealing. As a result, there occurs expansion or contraction in the DNA strand. During stalling of DNA polymerase, random point mutations occur. This leads to InDel mutations causing frameshift. In Mycoplasma bovis, in type III restriction modification system’s methyl-transferase mod gene there is an SSR of AG-repeats. With this gene mod, the gene res is present in the same open-reading frame (ORF) which has variable expression patterns owing to the presence of AG-repeats in the preceding gene mod. We studied patterns of slippage at AG-repeats, AT-repeats and G-repeats inserting them in SSR-part of mod (call mod’) by in vitro experiments. We found the threshold for slippage remains very close and it depends on the number of nucleotides in the entire stretch for Pfu DNA polymerase. For AG-repeats it is (AG)6, for other constructs as well it has been 12 nucleotide-long stretch of SSR.