Uniform TitleCBX2 and DNA damage repair: development of CBX2-specific reagents
NamePiso, Katherine (author), Ganesan, Shridar (chair), Glod, John (internal member), Banerjee, Debabrata (internal member), Rutgers University, Graduate School - New Brunswick,
SubjectMicrobiology and Molecular Genetics,
DescriptionDNA damage is inevitable, however, methods of DNA repair exist which allows cells to recover, or die, depending on the severity of the damage. Homologous recombination (HR) is one type of DNA damage repair that has the ability to repair double-stranded breaks (DSBs) in a high-fidelity manner. Unrepaired DSBs can led to cell death or genomic instability.
BRCA1is a tumor suppressor protein that is involved in the cellular response to DNA damage. Studies show that BRCA1 accumulates in S and G2 phases of the cell cycle, and after DNA damage re-localizes to nuclear repair foci. Cells that lack BRCA1 are unable to repair DNA using HR, and therefore tend to use more error-prone mechanisms.
A human auto-antisera, PIKA, first characterized in 2003 by William Earnshaw et al., recognizes proteins which contain chromodomains, and co-localize with BRCA1, before and after DNA damage. Chromodomains are critical features of the HP1 group of proteins, and the polycomb group (PcG) proteins. Prior work has suggested that the protein being recognized by the PIKA anti-sera, was CBX2, a member of the PcG and the polycomb repressive complex 1 (PRC1).
Unfortunately, the antibodies commercially available for CBX2 do not recognize both isoforms of CBX2, the truncated and the full-length. The purpose of this project was to generate plasmid constructs containing coding sequences for the CBX2 full-length protein using the directional cloning technique. A vector encoding a GST-CBX2 SL fusion protein was successfully generated, and conditions for expression and extraction of this protein from bacterial culture were optimized. Progress has been made in isolating a cDNA for the full length CBX2 isoform to generate both GST-fusion proteins and a GFP-tagged protein. By generating a proper CBX2 antibody for the full-length protein, we hope to show that this protein is involved in the normal response to DNA damage.
NoteIncludes bibliographical references (p. 46-48).
CollectionGraduate School - New Brunswick Electronic Theses and Dissertations
Organization NameRutgers, The State University of New Jersey
RightsThe author owns the copyright to this work.