Bio366-Test 6-Fall2009

Bio366-Test 6-Fall2009 - Bio-366 Test-6 November 5, 2009...

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1 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.
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2 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
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3 of the host can cause recombination between the head-to-tail copies of the transposon to leave a single copy at the target site (simple insertion). Some transposons carry their own resolution systems, for example, Tn3. The Tn3 resolvase is a site-specific recombinase that acts on its target sites to cause DNA excision or resolution. The action of the enzyme on the target sites present in the head-to-tail copies of the transposon within the cointegrate intermediate leaves one copy of the transposon at the insertion site. II
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This note was uploaded on 02/21/2010 for the course BIO 45 taught by Professor Sadasivan during the Spring '08 term at University of Texas at Austin.

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Bio366-Test 6-Fall2009 - Bio-366 Test-6 November 5, 2009...

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