Screening And Validation Of Proteins Interacting With Mre11

The cellular genomes are subject to various types of exogenous or endogenous DNA-damaging agents that lead to changes in chromosome structure and DNA damage. If the DNA double strand break can not be repaired effectively and timely, that will lead to many human disorders. In the long-term evolution, the organism has developed a series of DNA damage repair mechanisms in response to DNA damage. MRN complex, including Mrell, NBS1 and RAD50, is one of the key factors involved in DNA damage repair. MRN complex participates in DNA damage repair, homologous recombination, non-homologous end joining, telomere structure maintenance, cell cycle checkpoint control, DNA replication and to maintain genomic stability. Therefore, it is important to study the functions of the MRN complex and ascertain the mechanism.In this paper, we used the N terminal 335 amino acid residues of Mre11 as a bait (Mre11N335), cDNA library of the Drosophila melanogaster 21 h embryo as a prey pool, to do the Yeast Two-Hybrid screening. We used CG1945 yeast strain as a host, SD-T-L-H-3-AT dropout medium as selection medium, to do the first screening,β-galactosidase activity is another selection marker to be detected. We restreaked positive colonies on SD-L-T-H-3-AT plates 3 times to allow loss of some of the AD/library plasmids while maintaining selective pressure on both the DNA-BD and AD vectors. The positive colonies still contain duplicated library plasmids. We extracted plasmids from yeast strain, PCR amplified inserted fragments, digested with Haelll to sort the colonies and eliminate duplicates. The screened plasmids were transformed into E.coli, and done PCR amplification, HaeⅢdigestion again to sort the colonies and eliminate duplicates. The screened positive colonies were sent to sequence, blasted with Drosophila cDNA sequence and selected the colonies that have the correct reading frame with the Drosophila gene. The positive colonies need to be retested. We did cotransfection in AH 109 yeast strain, or yeast mating by AH 109 and Y187 yeast strains, and frameshift mutation to retest the interaction in vivo. We got five interacting proteins, CG4035 (eIF-4E), CG1216 (mri), CG4916 (me31B), CG5468 (TweedleM) and CG3140 (Adk2) through above experiments. The interaction was also proved by co-immunoprecipitation in vitro. We have demonstrated the interaction between CG1216 (mri), CG4916 (me31B) or CG3140 (Adk2)andMre11N335.