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Unformatted text preview: Bio-366 Test-6 November 5, 2009 Allowed time: 11.00 am to 12.10pm (I hr and 10 min) Name: UTEID: 1. Please be brief and to the point. I A . What is the chemical nature of the DNA cleavage reaction mediated by the transposase enzyme? What are the chemical groups left at the ends of the transposon and the adjacent ‘donor’ DNA as a result of strand cutting? Is the energy of the phosphodiester bond conserved during this reaction? The transposase enzyme mediates a hydrolytic reaction at the end of the transposon. This causes the formation of a 3’-OH at the transposon end and a 5’-phopshate adjacent to it in the donor DNA. No, the energy of the phosphodiester bond is lost during the hydrolytic step. B . What is the nucleophile employed during the strand joining step of DNA transposition? What is the chemical mechanism of this reaction? Is the energy of the target phosphodiester bond conserved during this reaction? The 3’-OH formed at the end of the transposon is the nucleophile in the strand joining reaction. The 3’-OH attacks the phosphodiester bond in the target DNA to form the phosphodiester junction between the transposon and the target. Since the reaction breaks one phosphodiester bond but simultaneously forms a second one, the reaction is transesterification. Yes, the energy of the phosphodiester bond is conserved during the strand joining reaction. C . For a DNA transposition event occurring by a replicative transposition mechanism, what is the intermediate formed? How many copies of the transposon are present in this intermediate? In what orientation are they present? What happens to the target sequence where the transposon is newly integrated? By what mechanism or mechanisms can the intermediate be resolved? In the figure, you can join A to B and C to D to represent replicative transposition from the donor circle (A- B) to the recipient circle (C-D). The intermediate formed during replicative transposition is called a ‘cointegrate’. In this intermediate, there are two copies of the transposon in head-to-tail orientation. The target sequence is duplicated. They are also in head-to- tail orientation. The two copies of the target sequence flank the cointegrate at the extreme ends, that is, they flank the left end of one transposon copy and the right end of the other. The cointegrate can be resolved by one of two mechanisms. The homologous recombination system ffd8ffe000104a46494600010201006d006d0000ffe20c584943435f50524f46494 c4500010100000c484c696e6f021000006d6e74725247422058595a2007ce000 20009000600310000616373704d534654000000004945432073524742000000 0000000000000000000000f6d6000100000000d32d4850202000000000000000 000000000000000000000000000000000000000000000000000000000000000 000000000000000001163707274000001500000003364657363000001840000 006c77747074000001f000000014626b707400000204000000147258595a0000 0218000000146758595a0000022c000000146258595a0000024000000014646d 6e640000025400000070646d6464000002c400000088767565640000034c0000...
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- Spring '08
- DNA, transposon end