How Cells Combat DNA Damage
In order to accurately replicate and pass on their genetic material, cells must repair DNA damage as it arises. Two of the most dangerous types of DNA damage are double-strand breaks and interstrand crosslinks. Failure to repair these lesions can result in cell death by apoptosis, while inaccurate repair can be mutagenic. Many human diseases, including Fanconi Anemia and other cancer-prone disorders, are caused by defects in repair mechanisms that deal with breaks and crosslinks. Our long-term goal is to determine how various DNA repair pathways are regulated during development and aging in a multicellular eukaryote.
Double-strand breaks can be repaired by two main classes of pathways: non-homologous end-joining and homologous recombination. Homologous recombination (HR) utilizes a homologous template for repair and is usually considered to be error-free. However, accumulating data suggests that HR can be mutagenic in certain contexts. We are testing the hypothesis that error-prone translesion DNA polymerases may be utilized during the initiation of repair synthesis during HR. We use site-specific double-strand break repair assays and other molecular genetic approaches in the model organism Drosophila melanogaster.
Figure 1. Male fly in which P-element induced double-strand breaks in eye progenitor cells are being repaired by homologous recombination (red patches) and non-homologous end-joining (yellow patches).
End joining represents a flexible set of mechanisms that can repair double-strand breaks when a homologous template is unavailable. Classical end joining (C-EJ) involves the protection, processing, and subsequent ligation of broken ends, and depends on the Ku70/80 heterodimer and the DNA ligase IV/XRCC4 complex. Cells lacking C-EJ proteins can utilize one or more alternative end-joining (alt-EJ) mechanisms. Currently, alt-EJ is poorly defined. In preliminary attempts to characterize these alt-EJ processes, we have found that Drosophila polymerase theta is involved in a particular form of alt-EJ that we have termed synthesis-dependent microhomology-mediated end joining. Interestingly, pol theta is also involved in DNA interstrand crosslink repair. We are further characterizing these dual roles of pol theta using biochemical and molecular biology approaches.
Figure 2. Two potential pathways of alternative end joining. (i) microhomology-mediated end joining, which involves annealing at pre-existing microhomologous sequences. (ii) synthesis-dependent microhomology-mediated end joining, which involves DNA synthesis and production of nascent microhomologies.
Our lab is particularly interested in the following projects:
- Investigating potential roles of translesion polymerases in DNA double-strand break repair.
- Determining the genetic components of alternative end-joining repair pathways.
- Using an inducible I-SceI endonuclease system to determine the extent that various repair pathways are utilized in different tissues and developmental stages.
- Identifying proteins and pathways that repair camptothecin-induced DNA damage.