Epigenetic modifiers play vital roles in directing DNA structure, regulating gene expression, and determining disease-states. Scientists have recently taken interest in a epigenetic marker, 5-hydroxymethylcytosine (5hmC), which represents less than one percent of cytosines in the mammalian genome. Initially, 5hmC was thought primarily to be an intermediate in methylcytosine metabolism, but now the field is beginning to recognize 5hmC's involvement in a variety of cellular functions, even calling it the sixth base of the genome. One observation is that high local genomic concentrations of 5hmC have been linked to DNA recombination. Furthermore, the recombination protein Endonuclease G (Endo G) has been recently found to preferentially cleave 5hmC modified DNA over unmodified DNA. The proposed research aims to explore this link between 5hmC, Endo G, and recombination. The hypothesis is that 5hmC-modified DNA forms unique hydrogen bonds that promote pausing of a migrating DNA Holliday junction, and the paused junction provides a stable substrate for Endo G to bind. This model will be explored from a structural perspective with two specific aims.
The first aim i s to determine the structure and thermodynamics of 5hmC DNA using x-ray crystallography and differential scanning calorimetry.
The second aim i s to define the role of 5hmC on Endo G recognition by testing the binding of Endo G to a series of 5hmC, 5methyl-C, and unmodified-C duplex and junction DNA constructs. Overall, the mechanisms behind recombination aren't fully understood, and 5hmC is likely an important part the regulation process. Since recombination is an integral part of the DNA damage repair mechanism, 5hmC may help to target local DNA regions for repair. Particular cell types, including cancer cells, express lower levels of 5hmC. This suggests 5hmC levels are linked to the disease-state of cancer cells by de-regulating their DNA repair. This research will help build the fundamental base of knowledge for understanding recombination-related diseases. A complete understanding of 5hmC will furthermore help decode the mechanisms of genomic regulation, impacting more than just the recombination field.

Public Health Relevance

I will study a new genetic marker, 5-hydroxymethylcytosine, which may help cells repair damaged DNA and prevent cancer. This marker helps by recruiting proteins that repair broken pieces of DNA using another DNA strand as a template. Without these repairs, the cell is more prone to becoming cancerous and diseased.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Predoctoral Individual National Research Service Award (F31)
Project #
1F31GM113580-01
Application #
8832524
Study Section
Special Emphasis Panel (ZRG1-F08-B (20))
Program Officer
Sledjeski, Darren D
Project Start
2015-01-01
Project End
2016-12-31
Budget Start
2015-01-01
Budget End
2015-12-31
Support Year
1
Fiscal Year
2015
Total Cost
$33,108
Indirect Cost
Name
Colorado State University-Fort Collins
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
785979618
City
Fort Collins
State
CO
Country
United States
Zip Code
80523
Vander Zanden, Crystal M; Rowe, Rhianon K; Broad, Amanda J et al. (2016) Effect of Hydroxymethylcytosine on the Structure and Stability of Holliday Junctions. Biochemistry 55:5781-5789
Scholfield, Matthew R; Ford, Melissa Coates; Vander Zanden, Crystal M et al. (2015) Force Field Model of Periodic Trends in Biomolecular Halogen Bonds. J Phys Chem B 119:9140-9