Single-molecule analysis of DNA polymerase in vitro and in vivo: a machine in action

Objective

DNA polymerases are dynamic molecular machines that faithfully copy genetic information during DNA replication and DNA repair. DNA polymerases use the information in a DNA template strand to synthesize a complementary copy by adding nucleotides to the 3 -end of a DNA primer. Extensive analysis has provided information on the structural organization of polymerases and led to proposals of a general scheme for nucleotide addition. However, there are many unanswered questions; we cannot really claim that we understand how the polymerase machine works in detail and how its function is modulated by its substrates, or by the intracellular environment of cells. One of the main reasons lies in the fact that crystal structures cannot directly capture the dynamics of multi-step processes, since the structures are static snapshots of structural states. Another limitation arises from the difficulty of interpreting biochemical studies of complex mechanisms, because of complications due to ensemble- and time-averaging of the observed signals.
We propose to overcome these limitations by studying DNA synthesis by a proofreading DNA polymerase (Pol I) through direct, real-time observation of its movements and interactions at the single-molecule level. We will study mechanisms of fidelity through comparisons involving mutant polymerases, mispaired nucleotides, or partially mispaired DNA, and make comparisons with RNA polymerases. We will also study the conformation and subcellular localisation of Pol I in bacterial cells. Our specific aims are:
1. To study conformational transitions of the DNA polymerase I fingers subdomain
2. To study translocational dynamics of DNA polymerase I on DNA substrates
3. To study the coupling of DNA polymerase I motions during nucleotide addition
4. To study conformational dynamics of DNA polymerase I during nick translation
5. To study the subcellular localization and conform

Fields of science (EuroSciVoc)

CORDIS classifies projects with EuroSciVoc, a multilingual taxonomy of fields of science, through a semi-automatic process based on NLP techniques. See: The European Science Vocabulary.

• engineering and technology materials engineering crystals
• natural sciences biological sciences genetics DNA
• natural sciences biological sciences genetics nucleotides
• natural sciences biological sciences genetics RNA

Programme(s)

Multi-annual funding programmes that define the EU’s priorities for research and innovation.

• FP7-IDEAS-ERC - Specific programme: "Ideas" implementing the Seventh Framework Programme of the European Community for research, technological development and demonstration activities (2007 to 2013)

Topic(s)

Calls for proposals are divided into topics. A topic defines a specific subject or area for which applicants can submit proposals. The description of a topic comprises its specific scope and the expected impact of the funded project.

• ERC-SG-ID1 - ERC Starting Grant Interdisciplinary Panel

Call for proposal

Procedure for inviting applicants to submit project proposals, with the aim of receiving EU funding.

ERC-2010-StG_20091118
See other projects for this call

Funding Scheme

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ERC-SG - ERC Starting Grant

Host institution

Beneficiaries (1)