Poster abstracts
Poster number 17 submitted by Zihao Qi
Structures of single-strand annealing protein and nucleoprotein complexes by native mass spectrometry
Zihao Qi (Department of Chemistry and Biochemistry, Resource for Native Mass Spectrometry Guided Structural Biology), Charles E. Bell (Department of Chemistry and Biochemistry, Department of Biological Chemistry and Pharmacology), Vicki H. Wysocki (Department of Chemistry and Biochemistry, Resource for Native Mass Spectrometry Guided Structural Biology)
Abstract:
DNA double strand breaks (DSB) severely damage genomic integrity, and if left unrepaired can cause diseases such as cancer. Homologous recombination (HR) and single strand annealing (SSA) are crucial pathways for repairing DSB. RecA/Rad51 family catalyzes strand invasion in HR, while non-RecA/Rad51 recombinase, also called single strand annealing protein (SSAP), can mediate HR and promote SSA. In our study, we seek to understand the oligomeric state and the DNA binding behavior of the SSAP family, specifically Rad52 and Mgm101 by native mass spectrometry (nMS) and surface induced dissociation (SID). nMS uses nano electrospray ionization (nESI) to transport biomolecules into the gas phase while retaining their non-covalent interactions. Surface induced dissociation (SID) is a gas phase activation method that breaks the weakest non-covalent interactions at low activation energies and can be used to measure oligomeric assembly and stability. With SID-nMS, we have found the Rad52-DNA binding domain (Rad52-DBD) is consistent with a ring-shaped 11-mer, which corresponds to the previous Cryo-EM result. Mgm101 is a Rad52-related SSAP from yeast mitochondria. With nMS, we found that Mgm101 forms a mixture of 18-mer and 19-mer, and the SID fragmentation suggests that it forms a ring-shaped structure.We investigated Rad52-DBD binding to DNA of different lengths, including 33-mer, 38-mer, 39-mer and 83-mer etc. We combined nMS with ion mobility (IM), which is a gas phase separation technique that can distinguish different conformations based on charge and shape. With IM, we can interrogate the conformational changes induced by DNA binding.Collectively, structures of different DNA binding states for SSAP have been evaluated, and new insights into DSB repair were obtained.
Keywords: Single strand annealing, Native mass spectrometry, Surface induced dissociation