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Focus on DNA damage and repair

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).

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, Bloom Syndrome, and other cancer-prone disorders, are caused by defects in repair mechanisms that deal with DNA breaks and crosslinks.

The McVey lab characterizes processes involved in repair of DNA breaks and crosslinks in the model metazoan, Drosophila melanogaster. Our long-term goal is to determine how various DNA repair pathways are regulated during development and aging in a multicellular eukaryote.

Our research employs a variety of genetic and molecular approaches, including powerful assays in which we can create double-strand breaks at known sites in the genome and recover and molecularly analyze repair events. The phenotypic results of one such assay are shown in Figure 1. Because Drosophila is a such a superb genetic system, we can use a wide array of tools to alter genes and gene expression patterns in order to test various DNA repair models.

Homologous recombination repair

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, experiments done using budding yeast suggest that HR can be mutagenic in certain contexts. We have demonstrated that error-prone, translesion polymerases, such as polymerase eta, polymerase zeta, and Rev1, have important roles during HR repair in flies (Kane et al., 2012). We are now testing the hypothesis that HR is also mutagenic in metazoans and that translesion DNA polymerases are responsible for at least a portion of this mutagenesis.

Non-homologous end joining

End joining represents a flexible set of mechanisms that can repair double-strand breaks when a homologous template is unavailable. Classical end joining (C-NHEJ) 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-NHEJ proteins can utilize one or more alternative end-joining (alt-EJ) mechanisms. Currently, alt-EJ is poorly defined.

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 (Yu et al., 2010; Chan et al., 2010, see Figure 2). 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. The relevance of our research to human health is highlighted by recent research showing that pol theta is highly expressed in several different types of human tumors and that overexpression correlates with poor prognosis. Could polymerase theta be a therapeutic target? We are currently pursuing this possibility.

Modeling genome instability-induced tumorigenesis

Recently, we have shown that flies lacking the Bloom Syndrome DNA helicase, BLM, develop gut and germline tumors early in their adult life (Garcia et al., 2011). Blm mutant flies also have a higher spontaneous mutation frequency and are prone to genome rearrangements and chromosome translocations. Because gut and germline cells continue to proliferate in adult flies, we hypothesize that genome rearrangements that occur in blm mutant adults activate tumor-promoting pathways. We are currently screening other candidate DNA repair mutants for tumor phenotypes.

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.