Genomic DNA is continually bombarded with a variety of insults, resulting in damage that must be repaired. By necessity, cells have evolved mechanisms to detect and repair broken strands of DNA, thereby preventing loss of important genetic information. Double-stranded DNA breaks (DSBs) are a type of damage that lead to particularly disastrous effects if not corrected. Homologous recombination (HR) is a highly conserved pathway that cells can use to repair DSBs. The consequences of disrupting HR are devastating. For example, mutations in the Rad51 recombinase are embryonic lethal in mice, and mutations in human Rad51 are linked to breast cancers. In addition, defects in BRCA2 account for at least 5% of all breast cancers and also confer a genetic predisposition to ovarian cancer. BRCA2 is thought to help regulate HR, and loss of this regulation may be the reason why this gene is linked to hereditary cancers. When a DSB occurs, the DNA ends are processed to generate 3'single-strand DNA (ssDNA) overhangs. The ssDNA ends then pair with homologous sequence elsewhere in the genome, and the missing DNA sequence is replaced using the homologous DNA as a template for replication. Finally, the replicated intermediate is resolved, regenerating the continuity of the broken DNA. While seemingly simple, HR requires the coordinated action of a complex repertoire of proteins, which are responsible for sensing damage, recruiting essential factors, and processing and repairing the damaged DNA. Our overall goals are to understand how these proteins sense and respond to damaged DNA. To help address this problem we have developed unique technologies that allow us to directly visualize hundreds of individual DNA molecules at the single molecule level using optical microscopy. Here we will try to determine how proteins process the ends of DNA at the early stages of HR, determine how these enzymes act on crowded substrates that reflect physiological settings, and determine how end processing is coupled to assembly of other protein complexes that are necessary to complete the repair pathway. We will reveal the spatial and temporal progression of these events by directly watching individual biochemical reactions in real time, and part of the significance of this project lies in the depth of the answers we strive to obtain.

Public Health Relevance

Homologs of RecBCD are found in ~90% of bacteria, and RecBCD is essential for triggering the SOS DNA damage response. When bacteria are taken up by phagocyctes they are subject to DNA damage due from reactive oxygen species (ROS) and they can also suffer DNA damage in response to a number of different antibiotics such as Ciprofloxacin. In the absence of RecBCD, bacteria have low viability when faced with DNA damage, but in the presence of RecBCD, they can remain viable by triggering the SOS response. As part of the SOS response, bacteria express mutagenic polymerases (e.g. pol IV &pol V), which are largely responsible for the ability of pathogenic bacteria to rapidly evolve drug resistance. Anti-bacterial drugs targets against RecBCD are expected to reduce the viability of bacteria in the face of our normal intracellular defenses and would also prevent the bacteria from developing drug resistance.

Agency
National Institute of Health (NIH)
Type
Research Project (R01)
Project #
5R01GM074739-08
Application #
8664873
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Janes, Daniel E
Project Start
Project End
Budget Start
Budget End
Support Year
8
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Columbia University (N.Y.)
Department
Biochemistry
Type
Schools of Medicine
DUNS #
City
New York
State
NY
Country
United States
Zip Code
10032
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Greene, Eric C (2016) DNA Sequence Alignment during Homologous Recombination. J Biol Chem 291:11572-80
Erdel, Fabian; Greene, Eric C (2016) Generalized nucleation and looping model for epigenetic memory of histone modifications. Proc Natl Acad Sci U S A 113:E4180-9
Greene, Eric C (2016) On the influence of protein-DNA register during homologous recombination. Cell Cycle 15:172-5
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Qi, Zhi; Redding, Sy; Lee, Ja Yil et al. (2015) DNA sequence alignment by microhomology sampling during homologous recombination. Cell 160:856-69
Lee, Ja Yil; Terakawa, Tsuyoshi; Qi, Zhi et al. (2015) DNA RECOMBINATION. Base triplet stepping by the Rad51/RecA family of recombinases. Science 349:977-81
Silverstein, Timothy D; Gibb, Bryan; Greene, Eric C (2014) Visualizing protein movement on DNA at the single-molecule level using DNA curtains. DNA Repair (Amst) 20:94-109
Collins, Bridget E; Ye, Ling F; Duzdevich, Daniel et al. (2014) DNA curtains: novel tools for imaging protein-nucleic acid interactions at the single-molecule level. Methods Cell Biol 123:217-34

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