Characterization of the N- terminal domain of Mre11 protein from rice (OsMre11) Oryza sativa.

Chromosomal breaks occur in the genome of all living organisms upon exposure to ionizing radiation, xenobiotics and as intermediates during normal cell cycle progression. Most of the information on DNA repair process has emerged from bacteria, human, mice, and yeast while information on plant DNA repair genes and proteins is limited. Among other DNA repair proteins, MRE11 forms the core of the MRN (Mre11-Rad50-Nbs1) complex and is the first responder to double strand breaks (DSBs), promotes repair either by Non-Homologous End Joining (NHEJ) or Homologous Recombination (HR). Till date, MRE11 has not been biochemically characterized from plant systems. Here, we report the in vitro biochemical activities of Oryza sativa MRE11. We cloned and purified the N- terminal region of OsMre11, which represents both the nuclease and DNA binding domains. The N- terminal end of OsMre11-N protein (∼55.0 kDa) showed binding activity with dsDNA, ssDNA and G-quadruplex DNA. Tryptophan fluorescence analysis also showed that OsMre11-N protein binds to ssDNA, dsDNA and G4 DNA in a protein concentration dependant manner. Additionally, OsMre11 protein showed exonuclease activity only in the presence of Mn2+. A protein concentration dependant endonuclease activity also was observed and was enhanced in the presence of Mn2+, Mg2+ and Ca2+. Put together, OsMre11 has properties similar to its counterparts in yeast and humans and may play an important role in cellular response to DNA damage in plants, especially rice.

C-terminal residues of rice translin are essential for octamer formation and nucleic acid binding.

Translin is a DNA/RNA binding protein involved in DNA repair and RNA metabolism. Previously, we had shown that rice translin (221 amino acids) exhibits biochemical activities similar to that of the human translin protein. Here we report the role of the C-terminal random coil in rice translin function by analyzing truncation (after 215th residue, Tra - 215) and substitution mutant proteins (Ser216Ala, Lys217Ala, Gln218Ala, Glu219Ala). Circular Dichroism (CD) analysis of Tra-215 showed deviations in comparison to Tra-WT. Truncation abolished the DNA binding activity and octamer formation as evidenced by the absence of ring like structures from TEM analysis. CD analysis of the substitution mutant proteins showed that the secondary structure was maintained in all the mutant proteins in comparison to wild type protein. Native PAGE and TEM analysis of the substitution mutants showed that Lys217Ala mutation completely abolished the octamer formation as rings and nucleic acid binding. Glu219Ala mutation also affected oligomerization but exhibited marginal RNA binding at higher protein concentrations and interestingly, failed to bind to DNA. However, Ser216Ala and Gln218Ala substitutions did not affect above mentioned activities of translin. Our results indicate that the C-terminal residues are one of the determinants of octamer formation in rice translin, with lysine at 217th position being the most important. Therefore, in conclusion, although the C-terminal residues do not form any defined secondary structure in the translin monomer, they are definitely involved in octamer formation and hence important for its molecular function. We have attempted to find the critical residues in translin function, which will advance our understanding of translin in DNA repair process in general and of rice translin in particular.

Biochemical characterization of plant Rad52 protein from rice (Oryza sativa).

DNA damage in living cells is repaired by two main pathways, homologous recombination (HR) and non-homologous end joining (NHEJ). Of all the genes promoting HR, Rad52 (Radiation sensitive 52) is an important gene which is found to be highly conserved across different species. It was believed that RAD52 is absent in plant systems until lately. However, recent genetic studies have shown the presence of RAD52 homologues in plants. Rad52 homologues in plant systems have not yet been characterized biochemically. In the current study, we bring out the biochemical properties of rice Rad52-2a protein. OsRad52-2a was over-expressed in Escherichia coli BL21 (DE3) cells and the protein was purified. The identity of purified OsRad52-2a protein was confirmed via peptide mass fingerprinting. Gel filtration and native PAGE analysis indicated that the OsRad52-2a protein in its native state probably formed an undecameric structure. Purified OsRad52-2a protein showed binding to single stranded DNA, double stranded DNA. Protein also mediated the renaturation of complementary single strands into duplex DNA in both agarose gel and FRET based assays. Put together, OsRad52-2a forms oligomeric structures and binds to ssDNA/dsDNA for mediating an important function like renaturation during homologous recombination. This study represents the first report on biochemical properties of OsRad52-2a protein from important crop like rice. This information will help in dissecting the recombination and repair machinery in plant systems.

Informed consent and subject motivation to participate in a large, population-based genomics study: the Marshfield Clinic Personalized Medicine Research Project.

BACKGROUND: The objective of this study was to measure subject perspective and reaction to participation in the Personalized Medicine Research Project (PMRP) and to identify factors predicting understanding of the study elements. METHOD: Self-administered questionnaires were mailed to 1,593 subjects (10% sample). The questionnaire had three sections: section A consisted of 21 factual questions; section B consisted of 14 questions to assess the level of understanding about the PMRP concepts, and section C asked about the purpose of the PMRP. RESULTS: The mean age of the 924 survey respondents was 52 years (SD = 16.9), with a range of 18-95 years. The majority of participants were female (n = 561, 61%). The percent of total correct responses for section A was significantly higher for females compared with males (males: 58.4% and females: 60.4%, t test = -2.18, p = 0.03) and age was significantly inversely related to percent of correct responses (beta coefficient = -0.122, p < 0.001). More than one third of the participants indicated that the USD 20 greatly influenced their decision to participate in the project. In a multiple logistic regression model, people living outside of Marshfield were significantly more likely to indicate that the USD 20 greatly influenced their decision to participate (odds ratio = 1.40, 95% confidence limit = 1.06, 1.86) and age was inversely related to the monetary influence on decision to participate (odds ratio = 0.98, 95% confidence limit = 0.97, 0.98). CONCLUSION: Future community consultation efforts should highlight areas of lower understanding. In addition, research coordinators may need to take more time informing males and older individuals about project details so that they are making truly informed decisions about study participation.