Studies on sister chromatid recombination in mammalian cells
By: by Andrea Jean Hartlerode
Series: Collections of the Harvard University Archives
Published: 2010
Language: eng
Number of pages: 217
Description: Failure to repair DNA double-strand breaks (DSBs), or their misrepair, may result in cell death or chromosomal rearrangements that can accelerate aging and promote carcinogenesis. To combat threats posed by DNA damage, cells have evolved two major pathways to specifically repair DSBs and thereby suppress genomic instability, homologous recombination (HR) and non-homologous end-joining (NHEJ). Early work raised the possibility that competition exists between the HR and NHEJ pathways, but other evidence exists pointing to collaboration between these pathways. To test whether NHEJ may operate in termination of sister chromatid recombination (SCR) events I examined the role of the classical NHEJ pathway in SCR. I find that deletion of XRCC4 does not abolish the use of error-prone NHEJ in the resolution of "early terminating" gene conversion events and that breakpoint junctions from both wildtype and XRCC4 null cells fall into two major classes: microhomology-mediated end-joining and insertions.

53BP1 is recruited to chromatin flanking a DSB and normally functions in XRCC4 -dependent NHEJ. The 53BP1 tandem Tudor repeat has been shown to interact in vitro with histone H4 dimethylated on lysine 20 (H4K20me2). To prove that 53BP1 -dependent DNA repair is mediated by a 53BP1-H4K20me2 interaction, I examined 53BP1 chromatin recruitment in cells lacking H4K20me2. I find that H4K20me2 is not absolutely required for the recruitment of 538P1 to chromatin, but that in the absence of H4K20me2, monomethylation of H4 on lysine 20 is required for this recruitment. Analysis of SCR at a molecular level in mammalian cells has been limited by difficulties achieving synchronized induction of a site-specific chromosomal DSB. To overcome this problem I developed an inducible site-specific DSB system based on the ligand-binding domain of the estrogen receptor. I used this system to demonstrate that a correlation exists between S phase and the amount of repair occurring by HR. In addition, I show that the major contribution of BRCA1 to HR occurs during S phase. This thesis expands the current knowledge of the mechanism of SCR in mammalian cells and focuses on SCR at various different levels to propel knowledge in the field outward in diverse directions.
Notes: "April 2010."

Thesis (Ph.D., Division of Medical Sciences (Experimental Pathology))--Harvard University, 2010.

Includes bibliographical references.