Transposition of maize Ac and Ds transposable elements in Saccharomyces cerevisiae and DNA repair of their excision sites

Jianhua Yu, Purdue University

Abstract

The first transposition system, Activator ( Ac) and Dissociation (Ds), was discovered genetically more than five decades ago in maize by McClintock and characterized molecularly in the early 1980s. However, the mechanisms for both transposition and repair of DNA damage at excision sites remain unclear. I address these questions by constructing and utilizing Ac/ Ds transposition in the yeast model genetic system. Results indicate that both the autonomous element Ac and non-autonomous element Ds transpose from yeast plasmids and chromosomes. Both Ac and Ds transpose more frequently from plasmids than from chromosomes and Ds transposes sooner than Ac. Hairpin intermediates form in adjacent host DNA during Ac/Ds transposition in yeast, similar to those formed during V(D)J rearrangement in vertebrates. Analysis of 172 independent, end-joining “footprints” formed after Ac/Ds excision in yeast indicates that 92% of them are microhomology-dependent. Just as in plants, a predominant Ac/Ds footprint is produced in yeast. The microhomology used in this predominant footprint is longer, and closer to the excision site than microhomologies used in less frequent footprints. In striking contrast to typical DNA double-strand breaks (DSBs) in yeast, more than half of the Ac excision sites are repaired by non-homologous end-joining (NHEJ) even when a homologous repair template is provided. I discuss a model in which the transposition complex may still bind to the broken host DNA ends after Ac/Ds move, and recruits host factors that target the repair of an excision site into the end-joining pathway. Interestingly, although previous data show that mre11-3, mre11-4 and mre11- 58S mutants can rescue the NHEJ ability of a mre11Δ mutant to repair DSBs, caused by HO endonuclease cutting, nearly to the level of wildtype, my data indicate that they can not rescue any ability to repair Ac excision sites. I propose that the Mre11 complex is involved in the opening of transposon-induced hairpins in yeast. Importantly, unlike the previous presumption of many investigators that DNA repair following Ac/ Ds excision is DSB repair, the evidence here suggests that these two types of DNA repair may be different. The difference is possibly due to that the transposition complex, including transposase, binds to DNA broken ends at Ac/Ds excision sites and that hairpin intermediates form in the host DNA during Ac/Ds transposition.

Degree

Ph.D.

Advisors

Weil, Purdue University.

Subject Area

Molecular biology|Genetics|Agronomy

COinS