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Analysis and confirmation of the results of a yeast two-hybrid screen carried out to identify proteins that interact with drosophila XRCC2

Author

Deepak Kumar, University of the Pacific

Date of Award

2005

Document Type

Thesis - Pacific Access Restricted

Degree Name

Master of Science (M.S.)

Department

Biological Sciences

First Advisor

Lisa Wrischnik

First Committee Member

Craig Vierra

Second Committee Member

Gregg Jongeward

Abstract

Repairing DNA damage is brought about by highly specific proteins that partake in a variety of DNA repair. Two of the most common types of damage are double-strand breaks (DSBs) and interstrand crosslinks. A single DSB or crosslink can potentially kill a cell if it is not repaired~ In human and other vertebrate cells, DSBs are repaired by two different mechanisms. The nonhomologous end-joining pathway can bring together the broken ends and join them, usually with the loss of some nucleotide sequence. A second pathway, homologous recombinational repair (HRR), is equally important. This repair process utilizes the information provided by another DNA molecule to restore damaged DNA. This molecule is usually a sister chromatid arising from DNA replication. This process is essentially error-free, unlike the end-joining process. Some HRR activity is required for proliferating cells to remain viable. The central protein player is RAD51, which with the help of other proteins such as XRCC2, XRCC3, RAD51B, RAD51C, and RAD51D, performs the critical initiating steps of homologous pairing and strand transfer. The proteins encoded by the familial breast cancer genes, brcal and brca2, also play an important role in HRR.

My project is concerned with studying proteins that interact with Drosophila melanogaster (XRCC2). Proteins interacting with DmXRCC2 were identified by using a yeast two hybrid system. "Bait fusion protein" (DmXRCC2 linked to GAIA BD) was constructed by Dr. Wrischnik. Tanya Dimetrijevich, a graduate student, used this bait to fish for interacting or "target" proteins. About 50 such proteins were found. I began validating these target proteins with the intention of exploring novel interactions and functions of DmXRCC2. The process of validating proteins interacting with DmXRCC2 yielded two very interesting candidate proteins-CaBPl and FAF. · CaBPl, also called protein disulfide isomerase P5, is an endoplasmic-reticulum calciumbinding protein. FAF belongs to a large family of deubiquitinating enzymes that cleave ubiquitin-protein bonds and play diverse roles in the ubiquitin pathway. One of the implications of such discoveries could be to compare and contrast DmXRCC2 and human XRCC2 in terms of their interactions and functions.

Pages

61

Recommended Citation

Kumar, Deepak. (2005). Analysis and confirmation of the results of a yeast two-hybrid screen carried out to identify proteins that interact with drosophila XRCC2. University of the Pacific, Thesis - Pacific Access Restricted. https://scholarlycommons.pacific.edu/uop_etds/622