Dealing with 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 lab’s long-term goal is to determine how various DNA repair pathways are regulated during development and aging in a multicellular eukaryote and how their mutagenic potential promotes genome evolution and genomic instability. To do this, we use a combination of genetic, genomic, molecular, and biochemical approaches in the model organism Drosophila melanogaster.
Figure 1. Mutagenic potential of various DNA double-strand break repair mechanisms. Top: non-homologous end-joining repair pathways; bottom: homologous recombination pathways. From Rodgers and McVey, 2016.
Mechanisms of alternative end joining
Double-strand breaks can be repaired by two main classes of pathways: non-homologous end-joining and homologous recombination. End joining represents a flexible set of mechanisms that can repair double-strand breaks when a homologous template is not available. Classical non-homologous 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 4 complex. Cells lacking C-NHEJ proteins can utilize one or more alternative end-joining (alt-EJ) mechanisms. We have shown that Drosophila DNA polymerase theta is important for alt-EJ, a function that other labs have shown is conserved throughout the metazoan lineage. Interestingly, Drosophila pol theta is also involved in DNA interstrand crosslink repair. We are further characterizing these dual roles of polymerase theta using biochemical and molecular biology approaches.
Figure 2. Models of polymerase theta-mediated alt-EJ. A double-strand break occurs (i) and DNA ends are resected (ii). Pol θ (green) aligns microhomologies (blue) located at the end of each ssDNA (iii). Pol θ synthesizes DNA to fill in the gap and strand displaces dsDNA, possibly aided by the helicase domain (purple) (iv). This repair process generates small deletions. Pol θ also aligns microhomologies that are located internally on ssDNA, leaving unpaired flaps (v). Flaps are cleaved by an endonuclease and Pol θ continues to synthesize DNA and displace dsDNA (vi). This process generates larger deletions. In the event that no microhomologies exist on ssDNA, Pol θ can utilize DNA overhangs as a template to generate microhomologies in “snap-back” synthesis, while displacing dsDNA (vii). Once microhomologies exist, they are aligned by Pol θ (viii) and Pol θ then fills in the gap (ix). This repair process generates templated insertions and deletions. From Beagan and McVey, 2016.
Mutagenesis during 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 are also investigating the roles of various DNA helicases in HR. Finally, we are collaborating with Catherine Freudenreich’s lab at Tufts to determine if trinucleotide repeat sequences may promote mutagenic HR.
Figure 3. Mechanisms that promote mutagenesis during homologous recombination. Interactions of the elements involved in mutagenic HR are indicated by arrows. Double arrows indicate that the elements influence each other. The propensity of each element to contribute to each type of mutation is designated in the outer circles. From McVey et al, 2016.
DNA damage tolerance in Drosophila
When replication forks arrive at sites of unrepaired DNA damage, damage tolerance mechanisms can promote bypass of the damage and allow for the continuation of replication. Tolerance mechanisms fall into two main classes: (1) translesion synthesis, when error-prone translesion DNA polymerases allow synthesis to proceed past the damage, and (2) template switching, when synthesis proceeds using an undamaged template (often a newly-synthesized sister chromatid). While the molecular signals that dictate which of these strategies is used are fairly well-understood in budding yeast, much less is known about DNA damage tolerance pathway choice in metazoans.
We have shown that the REV1 translesion polymerase is crucial for tolerance to methane methylsulfonate (MMS)-induced damage in Drosophila. Current efforts in the lab are focused on elucidating how REV1 promotes tolerance and with what proteins it interacts during bypass/repair of alkylation damage.
Figure 4. High levels of DNA double-strand breaks in rev1 mutant wing imaginal discs. Blue staining (DAPI) shows individual cells in each disc. Pink staining shows gamma-H2Av staining, which is a marker of DNA double-strand breaks. Shown are representative discs from flies lacking the entire REV1 protein (rev1 mutants), the carboxy-terminal domain of REV1 that interacts with other translesion polymerases (rev1-CTD), or the CTD and the SHPRH protein that is thought to be involved in template switching (rev1-CTD, shprh). The similar phenotypes of rev1 and rev1-CTD, shprh mutants suggests that REV1 might be involved in both the translesion synthesis and template switching pathways of damage tolerance.