Title

Rad51 Paralogs and Complexes – A Study of Protein Function and Interactions

Poster Number

41

Lead Author Major

Biochemistry

Format

Poster Presentation

Faculty Mentor Name

Joanna Albala

Faculty Mentor Department

Biological Sciences

Abstract/Artist Statement

Double-strand breaks in DNA have potential to result in cancer. Currently, there are two methods known for repairing such breaks and maintaining genome integrity. These are non-homologous end-joining (NHEJ) and homologous recombination repair (HRR). HRR is accomplished through the use of homologous DNA strands, one strand acting as a template to repair the other strand. A key protein in the strand exchange and homologous pairing used during HRR is Rad51. Presently, there are five human proteins with homology to Rad51, these paralogs are Rad51B, Rad51C, Rad51D, Xrcc2, and Xrcc3. Loss of these protein functions leads to chromosomal instability. In this research, the interactions of the five paralogs and the two complexes, both containing Rad51C that form from them will be investigated. Escherichia coli containing either human Rad51B or human Xrcc3 were grown and the plasmids containing the appropriate target gene were isolated. The plasmids were treated with restriction enzymes and the samples were run on an agarose gel to verify that the DNA of each repair gene was the expected size. Samples will be sequenced to verify the cDNA is mutation free. The target gene will be isolated and placed in a plasmid for expression in the yeast, Pichia pastoris, more suited for recombinant protein expression of higher eukaryotic organisms. The plasmid will be inserted into P. pastoris using electroporation and the corresponding protein will be expressed. These proteins will be used to examine protein interaction and function. This study will increase current understanding of Rad51C complex formation and function in the repair of DNA double-strand breaks.

Location

Grave Covell

Start Date

21-4-2012 10:00 AM

End Date

21-4-2012 12:00 PM

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Apr 21st, 10:00 AM Apr 21st, 12:00 PM

Rad51 Paralogs and Complexes – A Study of Protein Function and Interactions

Grave Covell

Double-strand breaks in DNA have potential to result in cancer. Currently, there are two methods known for repairing such breaks and maintaining genome integrity. These are non-homologous end-joining (NHEJ) and homologous recombination repair (HRR). HRR is accomplished through the use of homologous DNA strands, one strand acting as a template to repair the other strand. A key protein in the strand exchange and homologous pairing used during HRR is Rad51. Presently, there are five human proteins with homology to Rad51, these paralogs are Rad51B, Rad51C, Rad51D, Xrcc2, and Xrcc3. Loss of these protein functions leads to chromosomal instability. In this research, the interactions of the five paralogs and the two complexes, both containing Rad51C that form from them will be investigated. Escherichia coli containing either human Rad51B or human Xrcc3 were grown and the plasmids containing the appropriate target gene were isolated. The plasmids were treated with restriction enzymes and the samples were run on an agarose gel to verify that the DNA of each repair gene was the expected size. Samples will be sequenced to verify the cDNA is mutation free. The target gene will be isolated and placed in a plasmid for expression in the yeast, Pichia pastoris, more suited for recombinant protein expression of higher eukaryotic organisms. The plasmid will be inserted into P. pastoris using electroporation and the corresponding protein will be expressed. These proteins will be used to examine protein interaction and function. This study will increase current understanding of Rad51C complex formation and function in the repair of DNA double-strand breaks.