Bio366-Test5-Fall2009 - Bio-366 Test-5 October 27, 2009...

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1 Bio-366 Test-5 October 27, 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 are the two families of Conservative Site-specific recombinases? Why are they called ‘conservative’? What are the active site nucleophiles used by members of each family for strand cutting? What is the chemical mechanism used by these recombinases to carry out strand cutting and strand joining? The two families are the tyrosine family (or the integrase family) and the serine family (invertase/resolvase family). They are called ‘conservative’ because they conserve the energy of the phosphodiester bond during recombination. These recombinases utilize a transesterification mechanism during the strand cutting and strand joining steps of recombination. During strand cleavage, the active site tyrosine or serine attacks the phosphodiester in DNA to form a 3’- phosphotyrosine or 5’- phopshoserine linkage between DNA and the recombinase. In the strand joining step, the 5’-OH or the 3’-OH formed during the cleavage step attacks the phosphotyrosine or phosphoserine bond, respectively, to reform the DNA phosphodiester bond. Hence the recombination reaction is completed without the need for energy input. B . What are the active site nucleophiles employed by the two families of site-specific recombinases? Describe the chemical nature of the DNA ends formed as a result of strand cleavage by a serine site-specific recombinase and a tyrosine site-specific recombinase.
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2 The active site nucleophile for the serine family is a serine residue and that for the tyrosine family is a tyrosine residue. The two families are named based on their active site nucleophiles . Strand cleavage by a serine site-specific recombinase produces a 5’-phosphoserine on one side of the strand nick and a 3’-hydroxyl on the other side. Strand cleavage by a tyrosine site-specific recombinase produces a 3’-phopshotyrosine on one end of the nick and a 5’-hydroxyl on the other side. C . What are the nucleophiles used during the strand joining steps of serine family and tyrosine family site-specific recombination? During strand joining by a serine site-specific recombinase, the nucleophile is a 3’- hydroxyl group (formed during strand cleavage). During strand joining by a tyrosine site-specific recombinase, the nucleophile is a 5’-
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Bio366-Test5-Fall2009 - Bio-366 Test-5 October 27, 2009...

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