Repair of Protein-linked DNA Double Strand Breaks: Biochemical Characterization Using Adenovirus as a Model System

Brandon Lamarche

Appointment Period: 2009-2011 / Grant Years: [25,26,27]

Whether generated by chemical/physical damage (ionizing radiation, select chemotherapy drugs, and metabolic byproduct reactive oxygen species) or programmed enzymatic activity (meiosis and V(D)J recombination) DNA double strand breaks (DSBs) differ from all other types of DNA lesions in that the sequence information requisite for guiding repair is no longer contained within a contiguous duplex molecule. An important subclass of DSBs are those in which the termini of the break are covalently bound to proteins. Because they inhibit topoisomerases after DNA their strand cleavage step but prior to strand relegation, chemotherapeutic drugs such as topotecan and etoposide generate protein-linked DSBs (PL-DSBs). Regardless of the context in which PL-DSBs occur, removal of the covalently attached protein is essential in order for them to be repaired (via homologous recombination or non-homologous end joining). Despite the ubiquity of PL-DSBs, the mechanism of protein removal is poorly understood.

As a simplified yet highly analogous model system for studying the repair of PL-DSBs, we have made use of human adenovirus (Ad), which primes its genome for replication using a dCMP moiety that is covalently bound to the serine side chain of the virally-encoded Terminal Protein (TP). Positioned opposite to dG in the complementary strand, this serine-dCMP serves as the 3’ primer terminus for synthesis of the Ad genome. Upon completion, the newly synthesized strand is analogous to any other DNA strand with the exception that its 5’-terminus is “blocked” via the covalent linkage to TP. Because this TP-bound Ad genome is an excellent analog of the PL-DSBs generated during chemotherapy and meiosis, we have employed it as a substrate for assays aimed at identifying and characterizing the protein(s) responsible for TP/Spo11/topoisomerase removal.

WT adenovirus encodes multiple proteins that cause either degradation or mislocalization of numerous host DNA repair factors. During infection with a mutant form of the virus lacking these genes, host repair factors remain intact and consequently the Ad genome is perceived to be a PL-DSB. This results in TP being cleaved from the viral genome and subsequently Ad genomes get ligated into linear concatemers. By immuno-precipitating TP from lysates of cells infected with this mutant Ad, and subsequently labeling the pulled down protein with the template independent terminal transferase and radioactive dNTP, we have demonstrated that when TP is release from the viral genome it is attached to a short oligonucleotide (suggesting a nuclease, rather than phosphodiesterase or protease, mechanism of TP removal). We have now demonstrated in vivo that TP is removed from the Ad genome via a mechanism involving both Mre11 and CtIP. We are now focused on dissecting the specific roles that Mre11 and CtIP play in this process.

Lamarche, BJ, Orazio, NI, Weitzman, MD (2010). The MRN complex in double-strand break repair and telomere maintenance. FEBS Letters 584, 3682-3695. PMID: 20655309; PMC2946096

Lamarche BJ, Orazio NI, Weitzman MD. The MRN complex in double-strand breakrepair and telomere maintenance. FEBS Lett. (2010) 584(17):3682-95. Epub2010 Jul 24. Review. PMID: 20655309; PMC2946096.

Lamarche, BJ, Weitzman, MD (2010). Human CtIP Catalyzes Nucleolytic Release of Proteins Covalently Bound to DNA Double Strand Break Termini. In preparation.

NOTE: When Dr. Weitzman’s lab moved to UPenn last year, candidate remained at the Salk Institute in Tony Hunter’s lab. Brandon Lamarche remains as an active participant in our TG meetings and activities.