Helleases are molecular motors that play critical roles in nucleic acid metabolism, including replication, repair, recombination, and transcription. As observed in a number of diseases resulting from mutations in helicase genes, helicases are important for maintenance of cellular functions. A feature of these diseases is an increase in malignancies as a result of genome instability resulting from unregulated recombination. Gram-positive bacteria contain an essential helicase, PcrA, which is involved in UV-damage DMA repair, plasmid rolling-circle replication, and unidentified essential cellular processes. Cells containing reduced PcrA levels are hyper-recombinogenic, consistent with PcrA regulating homologous recombination. Due to its small size, ~700 residues, and likely monomeric state, PcrA is an ideal model for understanding helicase function and diseases arising from irregular helicase activities. PcrA possesses helicase activities and translocates along ssDNA. PcrA has four structural domains, 1 A, 1B, 2A and 2B, and likely unwinds dsDNA by an active mechanism that involves enzyme inchworming. The 2B domain, which interacts with dsDNA, has been hypothesized to be critical for the conformational changes in PcrA during DMA unwinding. Using single-pair fluorescence resonance energy transfer (spFRET), magnetic tweezers and rotor beads, I propose to correlate the conformational changes occurring within individual PcrA molecules with their translocation and DMA unwindase activities. I will monitor conformational changes using spFRET and test the inchworm hypothesis of PcrA translocation, follow the mechanical rotation of the DMA labeled with a rotor bead as an immobilized PcrA molecule unwinds it; these rotations should correlate with the number of base pairs unwound. Combining spFRET with rotation, I propose to correlate the intramolecular movements of PcrA with the unwinding of DNA at the level of individual base pairs. This correlation will allow for a precise determination of the step size of PcrA and a better understanding of the relationship between helicase dynamics and activity. Using designed di-cysteine bonds, I will limit the dynamics of the 2B domain and monitor the effects of these constraints on PcrA translocation and unwinding activities. Lay Summary: Helicases have a large influence on the maintenance of life, as observed in a number of human diseases resulting from mutations in helicase genes. These diseases have an increase in malignancies due to genome instability resulting from unregulated recombination. Due to its small size, ~700 residues, and likely monomeric state, PcrA is an ideal model for furthering the understanding of helicase functions and malignancies arising from irregular helicase activities. ? ? ?
| Fagerburg, Matt V; Schauer, Grant D; Thickman, Karen R et al. (2012) PcrA-mediated disruption of RecA nucleoprotein filaments--essential role of the ATPase activity of RecA. Nucleic Acids Res 40:8416-24 |
