Functional analysis of the bacterial DNA repair protein complex SbcCD

Saathoff, Jan-Hinnerk Friedrich Fokke (2018): Functional analysis of the bacterial DNA repair protein complex SbcCD. Dissertation, LMU München: Fakultät für Chemie und Pharmazie

Abstract

The genomic integrity of all organisms is constantly challenged by genotoxic stress originating from endogenous and exogenous sources, with stalled replication forks (RFs) and double-strand breaks (DSBs) being among the most deleterious forms of DNA damage. Failure to properly respond to genomic distress can be highly mutagenic and lead to chromosomal aberrations. Thus, cells have evolved distinct DNA repair mechanisms. The Mre11-Rad50-Nbs1 (MRN) complex holds a key position in the DNA damage response (DDR) and is involved in the repair of DSBs, stalled RFs and dysfunctional telomeres. MRN acts by sensing and processing these diverse DNA structures and mediates signaling via the kinases ATM and ATR in eukaryotes. Although the MRN complex has been intensively studied for the last two decades, its fundamental mechanisms of action are still poorly understood. In particular, the nature of its ATP-dependent nuclease activities and how it specifically recognizes DNA ends remains unknown. The aim of this work was to investigate the biochemical activities of the bacterial Mre11-Rad50 homolog, SbcCD. For this purpose, biochemical assays were developed and established to study and functionally connect the enzymatic activities of SbcCD. These assays showed that SbcCD has a low basal ATPase rate. ATP hydrolysis is increasingly stimulated by (i) supercoiled DNA, (ii) double-stranded DNA and (iii) DNA ends. For its nuclease activity, SbcCD strictly requires DNA ends. SbcCD’s exonuclease activity depends on ATP binding, whilst the endonuclease activity requires ATP hydrolysis. A protein-blocked DNA end stimulates SbcCD’s endonuclease, which leads to internal cleavage of both DNA strands approximately 25 base pairs from the DNA end. Upon ATP hydrolysis, SbcCD also distorts the internal structure of the DNA duplex, implying that its DNA melting and endonuclease activities are functionally coupled. The position of SbcCD’s endonucleolytic cleavage is sensitive to 5 nucleotide DNA bubbles, which are cleaved on the 5’ side of the bubble. Therefore, a DNA bubble could be a transient intermediate, which is required for endonucleolytic cleavage. To generate this intermediate, it appears that a native SbcD dimer interface and the plasticity of the dimer interface are important. SbcCD cleaves the scissile phosphate on different sides, producing either 3’ or 5’ phosphates. The distinct cleavage products are determined by both the nuclease activity itself and the strand polarity. This suggests that the exo- and endonuclease activities have distinct cleavage mechanisms and could involve a geometrically flipped SbcCD complex. The presented results provide not only a more detailed knowledge of the mechanochemical performance of the SbcCD’s enzymatic activities, but also provide an important foundation for future structural investigations.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Themengebiete: 500 Naturwissenschaften und Mathematik ; 500 Naturwissenschaften und Mathematik > 540 Chemie
• Fakultäten: Fakultät für Chemie und Pharmazie
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 10. Juli 2018
• 1. Bericht­erstatter:in: Hopfner, Karl-Peter