The goal of the present proposal is to characterize the structure and assembly dynamics of the prokaryotic actin-like protein, ParM, and to understand how the biochemical properties of ParM and its regulators affect the ability of ParM filaments to find cargo molecules and actively push them to the poles of rod-shaped cells. For these studies we will combine three approaches: (i) in vitro biochemical and biophysical assays on ParM and its regulator the ParR/parC complex, (ii) reconstituted motility assays, and (iii) in vivo studies of filament assembly and cargo movement. ? ? The parM gene is part of a partitioning locus (the par operon) found on many low-copy plasmids, such as the R1 and R100 drug-resistance plasmids. To ensure that a copy of the plasmid is inherited by each daughter during cell division, the par operon constructs a simple bipolar spindle from three components: (1) a stretch of centromeric DNA called parC (Dam et al., 1994); (2) a repressor protein, ParR, that binds the parC locus; and (3) the actin-like protein ParM (van den Ent et al., 2002). The ParR/parC complex is thought to harness the force of ParM polymerization to push plasmids through the cytoplasm (Moller-Jensen et al., 2002 and Moller-Jensen et al., 2003). We previously characterized the assembly dynamics of ParM filaments (Garner et al., 2004) and, more recently, reconstituted ParM-based DNA segregation in vitro using purified components (Preliminary Results). To our knowledge, this is the first such reconstitution of any biological system for segregating DNA and it provides a unique opportunity to understand how prokaryotes use cytoskeletal systems to organize their intracellular spaces. For the present study, we propose the following specific aims: ? ? 1. Determine the structural and biochemical bases of ParM assembly dynamics. ? 2. Determine the molecular mechanism by which the ParR/parC complex promotes ParM polymerization. ? 3. Determine the effect of perturbing the biochemical properties of ParM and the ParR/parC complex on the accuracy and efficiency of plasmid segregation in vivo and in vitro. ? ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM079556-03
Application #
7488780
Study Section
Special Emphasis Panel (ZRG1-IDM-H (03))
Program Officer
Deatherage, James F
Project Start
2006-09-25
Project End
2010-08-31
Budget Start
2008-09-01
Budget End
2009-08-31
Support Year
3
Fiscal Year
2008
Total Cost
$257,056
Indirect Cost
Name
University of California San Francisco
Department
Pharmacology
Type
Schools of Medicine
DUNS #
094878337
City
San Francisco
State
CA
Country
United States
Zip Code
94143
Belin, Brittany J; Lee, Terri; Mullins, R Dyche (2015) DNA damage induces nuclear actin filament assembly by Formin -2 and Spire-½ that promotes efficient DNA repair. [corrected]. Elife 4:e07735
Polka, Jessica K; Kollman, Justin M; Mullins, R Dyche (2014) Accessory factors promote AlfA-dependent plasmid segregation by regulating filament nucleation, disassembly, and bundling. Proc Natl Acad Sci U S A 111:2176-81
Petek, Natalie A; Mullins, R Dyche (2014) Bacterial actin-like proteins: purification and characterization of self-assembly properties. Methods Enzymol 540:19-34
Belin, Brittany J; Goins, Lauren M; Mullins, R Dyche (2014) Comparative analysis of tools for live cell imaging of actin network architecture. Bioarchitecture 4:189-202
Mullins, R Dyche; Hansen, Scott D (2013) In vitro studies of actin filament and network dynamics. Curr Opin Cell Biol 25:6-13
Vale, Ronald D; DeRisi, Joseph; Phillips, Rob et al. (2012) Graduate education. Interdisciplinary graduate training in teaching labs. Science 338:1542-3
Polka, Jessica K; Kollman, Justin M; Agard, David A et al. (2009) The structure and assembly dynamics of plasmid actin AlfA imply a novel mechanism of DNA segregation. J Bacteriol 191:6219-30
Choi, Charina L; Claridge, Shelley A; Garner, Ethan C et al. (2008) Protein-nanocrystal conjugates support a single filament polymerization model in R1 plasmid segregation. J Biol Chem 283:28081-6