The G·T mismatch
The first difference is characterized in that it is a mismatch G · T (by X-ray crystallography and NMR) double-stranded DNA. codon tRNA: the anticodon interaction G · T, wobble accept the settings of the type proposed by pairing erroneous description G · U the Brenner and click on the codon position of third in the mRNA to first base pairs
Mismatch endonuclease of VSR recognizes (T or W = A) Nick CTWGG sequence and underlined thymine contact guanine of DNA backbone on the 5 ‘end of the mispaired thymine. Using the nucleotide analogs and T G, we examined the functional groups of the pair mismatches that are recognized by the enzyme. For example, guanine, 2 removal of the methyl group, – – thymine, 5 to 60% reduction of the activity of such removal of the amino group, reducing cleavage by 90%. 2 – When you place a pair Neburarin or T amino purine, it generates a mismatch that are cut low rate (0.1%) much. If the base is removed, to generate (1 ‘, 2’-deoxy ribose) pseudoabasic the space, the enzyme produces a site-specific cleavage, but still, only 1% of the initial rate. CT mismatch and TT is disconnected low price (~ 1%) in this position, but Watson (for example, AC and GA) and other bases – mismatch between Crick base pairs, is not cleaved by the enzyme . There is no cut that can not be replaced by the mismatch difluoro-T.
In E. coli, I catalyzes the methylation of cytosine residues in the (T or W = A) sequence CCWGG underlined the DCM protein. 5 – tends to hydrolyze deamination producing thymine methylcytosine, discrepancies GT, if this situation does not repair may be due to causes transition mutation. Depends on Paula and gene VSR, it is stimulated by MutS with Mutl strong, GT mismatch due to this mechanism is corrected by (patch DNA repair synthesis very short) time to repair VSP. The VSR gene product, a DNA mismatch endonuclease that nickname double-stranded DNA sequence CTWGG. Leaving a 5′-phosphate group of the thymidine and cut it at the 5 ‘end of the thymidine residues underlined in mismatch 2’-deoxyguanosine. Cut is a thing of the field-specific sequence-dependent and-independent. As NTWGG and CTWGN, GT mismatch repair activity of E. coli endonuclease VSR examined the context array of different positions of one or two different from the array penta standard. All, some at a rate low, but I was treated by the enzyme.
I had the structure of the enzyme bound to oligonucleotide crystal structure of the protein is determined, the VSR disconnected. Aromatic residues of three, the crystal structure reveals a new authentication mechanism that is inserted into the DNA major groove deformation stacking local base pairs. Contact mismatch guanine and Lys89 in the main chain carbonyl of Met14 minor groove within the main groove, it while contacting only with thymine, which can be distinguished from cytosine made by Asn93. Some specific recognition of little mismatched base pairs, but has good spatial matching with the protein surface. Watson Standard – Thr19 disposed in the auxiliary groove which prevents interaction with Crick base pairing, however, it is possible to connect to the GT by thymine is shifted toward the major groove of wobble base pairs. The Phe67 stack the exposed portion of the mismatch thymine in the main groove directly.
Since a two-component natural DNA, GT mismatch lacks a marker to identify the DNA strands damaged. It was used a basis for some similar to explore the role of different functional groups GT mismatch recognition by VSR endonuclease in this study. There are structural studies GT contradictions some may form a pair of amino groups to guanine is known – two wobble base. Guanine which can form a base pair such investigated the importance of the first one with a substituent of inosine, but we are not exposed to the amino group. It is not possible in contrast to aminopurine and holds the amino group, it is paired with thymine change – 2. Neburarin lacks one of these functional groups. The role of various groups of the thymine residue (No 5 – methyl) deoxyuridine hydrogen bond donors, such as thymine and the same receptor, the possibility of forming a pair of wobble bases according to a thin configuration and It is, are those showing the arrangement, 8 – this G. that was confirmed at oxoguanine, to be able to correct the mismatch GT and VSP repair GU, has been shown previously. Further, by replacing the T or G and abasic site, discussed the importance of the base.
Complete details for the purification and cloning of endonuclease VSR will be published elsewhere. In a nutshell, VSR gene is prepared by PCR synthesis of the 50mer oligonucleotide according to the method of stemmer like. Generating a five VSR plasmid The PCR fragment was cloned between the BamHI and Ndel from the plasmid pET15b site. VSR endonuclease was purified by Ni-NTA resin and expressed as a His-tagged protein. Is determined by SDS-PAGE proteins were purified and was estimated by staining with Coomassie blue> to be 95% pure. Protein concentration is determined using the method of von Hippel and Gill A280 was estimated using an extinction coefficient of 31,010 M-1 -1. I was stored at -20 ° C in glycerol 15% dithiothreitol 7.6,1 mM NaOH buffer pH, including NaCl, 50mM HEPES / 500mM, EDTA of 1mM, at a concentration of 25μM it.