View Program - 63rd Annual Drosophila Research Conference

DNA double-strand breaks can be repaired using several different repair pathways. These include homologous recombination, classical-non homologous end joining, and a third, less well-understood set of mechanisms known as alternative end-joining. Alternative end joining repair is extremely inaccurate, causing insertions, deletions, and/or indels and has been linked to several different types of cancers. One type of alternative end joining, known as synthesis-dependent microhomology mediated end-joining (SD-MMEJ), occurs when short repeated sequences near the break site form secondary structures that prime nascent synthesis of microhomologies that are then used to repair the DNA.
In yeast, the replication protein A (RPA) heterotrimer has been shown to prevent another similar alternative end-joining pathway, microhomology mediated end-joining. To bind efficiently, RPA requires approximately 30 base pairs of single stranded DNA. Previous data from our lab demonstrate that movement of the short-repeated sequences to a distance greater than 30 bp from the break site reduces the frequency of SD-MMEJ usage from 15% to <1%. Therefore, we hypothesize that RPA that may inhibit SD-MMEJ by binding to short stretches of single stranded DNA and preventing secondary structures from forming. To test this, we have obtained a fly stock with a P element inserted in the 5’ UTR of the RPA70 gene, which severely decreases RPA70 expression. Cas9-induced breaks will be generated in a set of reporter plasmids that are injected into embryos from this stock. Deep sequencing and computational analysis of the inaccurate repair products will allow us to discern the role of RPA in SD-MMEJ repair. In addition, these studies may shed light on alternative end joining repair processes in cancer cells, which likely experience RPA exhaustion due to the accumulation of single-stranded DNA during perturbed DNA replication.

Alternative End Joining Preferences in RPA-Deficient Drosophila