Single-Molecule Assembly of Protein-DNA Complexes

  • Kowalczykowski, Stephen C, (PI)

Project: Research project

Project Details

Description

DESCRIPTION (provided by applicant): The major objective of this grant proposal
is to develop a new experimental technique for the study of protein-DNA
interactions at the single-molecule level. This proposal describes a novel
instrument to study the dynamics and function of individual complexes of
proteins and DNA. The design will permit the direct visualization by
fluorescence microscopy, in real-time, of the assembly, disassembly, and
movement of proteins on single, optically-trapped DNA molecules using a novel,
multi-port, laminar-flow, micro machined flow-cell. This instrument will allow
us to readily introduce an individual, optically-trapped DNA molecule (or
protein-DNA complex) sequentially to other proteins and/or reaction conditions,
and visualize the changes in structure/assembly of the molecules in real-time
using multi-wavelength fluorescence microscopy. The instrument will also
measure the forces generated by these protein-DNA interactions.
Several different protein-DNA complexes will be examined; each is an essential
component of genetic recombination. Two classes of proteins that will be
examined are the DNA motor proteins known as helicases, and the DNA strand
exchange proteins known as recombinases. The DNA helicases central to this
process in Escherichia coli are the RecBCD enzyme and the RecQ helicase. The
recombinases essential to this process are RecA (prokaryotes) and Rad51
(eukaryotes) proteins, and their ancillary protein components. This grant
proposal has two specific aims. The first is to develop an instrument that will
permit analysis of individual assemblies of protein-DNA complexes. The second
broad objective is to examine the formation, dissociation, and translocation of
the several specific DNA helicases (motor proteins) and DNA-enzyme complexes
(protein machines) that are involved in genetic recombination and DNA repair.
Development of this instrument will enable new types of single-molecule
experiments, experiments that will uncover information about the dynamics and
function of protein-DNA interactions that cannot be obtained from large
ensembles of DNA molecules.
StatusActive
Effective start/end date7/15/023/31/20

Funding

  • National Institutes of Health: $258,887.00
  • National Institutes of Health: $315,700.00
  • National Institutes of Health: $314,675.00
  • National Institutes of Health: $310,338.00
  • National Institutes of Health: $285,912.00
  • National Institutes of Health: $288,800.00
  • National Institutes of Health: $303,661.00
  • National Institutes of Health: $288,800.00
  • National Institutes of Health: $99,697.00
  • National Institutes of Health: $314,675.00
  • National Institutes of Health: $409,090.00
  • National Institutes of Health: $97,346.00
  • National Institutes of Health: $97,346.00
  • National Institutes of Health: $288,800.00

ASJC

  • Medicine(all)
  • Biochemistry, Genetics and Molecular Biology(all)

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