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Scientific Presentations Summer 2009 - Dana-Farber/Harvard ...

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Determining the Mechanisms of DNA Double-Strand Break in<br />

Heterochromatin & Euchromatin<br />

Raquel L. Graham<br />

Mentor: Peter O’Donovan, PhD<br />

<strong>Scientific</strong> Advisor: David Livingston, MD<br />

<strong>Dana</strong>-<strong>Farber</strong> Cancer Institute<br />

Our genetic material DNA resides in a chromatin structure composed of DNA and<br />

histone proteins. There are two forms of Chromatin—heterochromatin and euchromatin.<br />

Heterochromatin is tightly compacted and transcriptionaly inactive, whereas<br />

euchromatin is less compacted and transcriptionaly active. Chromatin impacts all<br />

DNA Metabolic processes, including DNA damage and DNA repair. The DNA double-strand<br />

break [DSB] is the most toxic of various types of DNA damage because<br />

both DNA strands are damaged. There are two major categories to repair DNA DSB:<br />

homologous recombination and non-homologous endjoining. Homologous recombination<br />

is error free and non-homologous endjoining is error prone. It has been<br />

suggested that there are differences in DNA repair between heterochromatin and euchromatin.<br />

The purpose of this study is to find out whether or not there are differences<br />

in mechanisms of DNA DSB repair of heterochromatin and euchromatin. To test<br />

this hypothesis, we have developed a system that allows us to generate a single DNA<br />

DSB in a cell. The DNA DSB will be either in heterochromatin or euchromatin. We<br />

will be using an enzyme called ISce-I and female mouse embryonic fibro-blast cells<br />

[MEFs]. A restriction site will be placed in the X Chromosome of the MEFs because<br />

the female has two X Chromosomes, active X (euchromatin) and inactive X (heterochromatin).<br />

Then ISce-I will be introduced into the nucleus to generate a DNA DSB<br />

in the X Chromosome. After DNA DSB repair, genomic DNA will be extracted from<br />

cells, PCR amplified, and sequenced for the region around DSB. The sequence will<br />

tell what repair mechanism was used for DSB, i.e. homologous recombination or<br />

non-homologous endjoining. Therefore we will be able to determine whether different<br />

repair mechanisms were used between heterochromatin vs. euchromatin.

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