Preliminary Structural Analyses Of Specific 5-hydroxymethylcytosine Binding Protein HMCES From ES Cell Of Homo Sapiens And SsDNA-binding Protein Rect From E.Coli

1. Preliminary structural analyses of specific 5-hydroxymethylcytosine binding protein HMCES from ES cell of Homo sapiensThe epigenetic changes or modifications including DNA methylation, histone modification, chromation remodeling, and the DNA methylation, and the DNA methylation is among the best studied epigenetic modifications. The 5-methylcytosine has been described as the fifth base in the genome and the oxidized base generated by the TET enzymes from 5-methylcytosine (5-hydroxymethylcytosine) is considered to be the sixth base of the genome of higher organism because of their important role in the.DNA methylation and demethylation process. HMCES can bind DNA containing the oxidized derivatives of 5-mC (especially 5hmC) and then recruit DNA repair enzymes that replace DNA containing these bases with unmodified bases. Its autoproteolytic activity is part of a functional switch.In this study, we have cloned the full length of HMCES gene in vitro and constructed the two expression vectors, HMCES and HMCES-MBP, and then we have expressed them with prokaryotic expression system. However, we found HMCES clearance after the TEV enzyme digestion, so we turned to optimize the expression of HMCES directly and uncovered the autoproteolytic activity of HMCES. We then found the truncated fragments (IIMCES-276) of HMCES with the methods of mass spectrometric analysis and Secondary structure prediction. We get the crystal of HMCES-276 after the clone, expression, purification and screen of it. The next task is uncovering the X-ray structure of HMCES and providing insights into its functions.2. Express and purify of the ssDNA-binding protein RecT from E.coliThe DNA molecule frequently suffers damage and changes due to the external environment and multi-factor in cell, in the long-term evolution, the cell have developed a series of DNA damage repair mechanisms in response to DNA damage to diminish the effect to cell, including photoreactivation repair, base excision repair, nucleotide excision repair, recombination repair, SOS repair, double strand break repair, etc. There are some differences in recombination repair pathway between eukaryotes and prokaryotes. The Rec proteins play a major role in recombination repair pathway in prokaryotes, including Rec A, RecBCD, RecFOR and RecET. RecT binds to single-stranded DNA and promotes the renaturation of complementary single-stranded DNA in E.coli interaction with RecE.In this part of work, we try to explore the structural basis of RecT promotes the renaturation of complementary single-stranded DNA via analysis of RecT structure. We have cloned the full length of RecT gene in vitro and constructed two expression vectors. We didn't get the crystal with the expression, purification and screen of RecT, so we try to optimize the process of screen, including adding Mg2+ and ssDNA. We changed our thought and focused on the fragment of RecT after we found the optimization didn't work after several weeks. We found there is a fragment (RecT) that we can get the protein with purifying and then we screened the crystal. The next task is ongoing.