Involvement of the nucleotide excision repair protein UvrA in instability of CAG-CTG repeat sequences in Escherichia coli

Several human genetic diseases have been associated with the genetic instab ility, specifically expansion, of trinucleotide repeat sequences such as (C TG)(n)(.)(CAG)(n). Molecular models of repeat instability imply replication slippage and the formation of loops and imperfect hairpins in single stran ds. Subsequently, these loops or hairpins may be recognized and processed b y DNA repair systems. To evaluate the potential role of nucleotide excision repair in repeat instability, we measured the rates of repeat deletion in wild type and excision repair-deficient Escherichia coli strains (using a g enetic assay for deletions). The rate of triplet repeat deletion decreased in an E. coli strain deficient in the damage recognition protein UvrA. More over, loops containing 23 CTG repeats were less efficiently excised from he teroduplex plasmids after their transformation into the uvrA(-) strain. As a result, an increased proportion of plasmids containing the full-length re peat were recovered after the replication of heteroduplex plasmids containi ng unrepaired loops. In biochemical experiments, UvrA bound to heteroduplex substrates containing repeat loops of 1, 2, or 17 CAG repeats with a Kd of about 10-20 nm, which is an affinity about 2 orders of magnitude higher th an that of UvrA bound to the control substrates containing (CTG)(n)(.)(CAG) (n) in the linear form. These results suggest that UvrA is involved in trip let repeat instability in cells. Specifically, UvrA may bind to loops forme d during replication slippage or in slipped strand DNA and initiate DNA rep air events that result in repeat deletion. These results imply a more compr ehensive role for UvrA, in addition to the recognition of DNA damage, in ma intaining the integrity of the genome.