Characterisation of the Protein Kinase Domain of the Large Subunit of Herpes Simplex Virus Ribonucleotide Reductase

Cooper, Jillian Katherine (1994) Characterisation of the Protein Kinase Domain of the Large Subunit of Herpes Simplex Virus Ribonucleotide Reductase. PhD thesis, University of Glasgow.

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Ribonucleotide reductase catalyses the reduction of ribonucleoside diphosphates to deoxyribonucleoside diphosphates, this enzyme plays a central role in nucleotide metabolism and is essential for DNA replication. Herpes simplex virus type 1 (HSV-1) ribonucleotide reductase is composed of two subunits, a 136kDa large subunit (R1) and a 36kDa small subunit (R2). The large subunits of the herpes simplex (type 1 and type2) members of the alphaherpesvirus family are unusual in that they have a 325 amino terminal extension which shows no homology to any other reductase large subunit sequenced (Nikas et al., 1986). Both HSV-1 R1(Conner et al., 1992b) and HSV-2 R1(Chung et al., 1989; Chung et al., 1990) autophosphorylate and in HSV-2 R1 the autophosphorylating activity maps to the unique amino terminal region (Luo et al., 1991; Ali et al., 1991). The herpes simplex viruses are similar in many aspects, their pathology is similar and both are neurotropic, becoming latent in sensory ganglia. These similarities are reflected in the conservation of genes between the two types. Primary effluences of corresponding all the HSV-1 genes has been determined (McGeoch et al., 1988) and corresponding in the HSV-2 genes sequenced a high degree of homology has been noted. R1 is unusual in that it shows a varying degree of conservation, with a carboxy terminal portion which is 80% conserved between HSV-1 and HSV-2 and an amino terminal region which shows only 38% conservation. The lack of conservation of this region raised the question of how the autophosphorylating/kinase activities of the two differ. To determine if this lack of conservation explains the differences that were observed in both the autophosphorylating and transphosphorylating activities of HSV-1 and HSV-2 R1, HSV-2 R1 was overexpressed in E.coli and purified to apparent homogeneity using the same expression system and purification scheme used for HSV-1 R1. Analysis of E.coli expressed HSV-2 R1 showed that it has properties similar to those described for HSV-1 R1 (Conneret al., 1992b). Autophosphorylation of HSV-2 R1 is similar to that of HSV-1 R1 in its stimulation by basic polypeptides. Like HSV-1 R1 the HSV-2 large subunit is susceptible to proteolytic cleavage. Proteolytic cleavage products of a similar Mwt are (Thserved in type 1 and type 2 R1 extracts. Previously it has been shown that portions of the removal portions of the amino terminal region of HSV-1 R1 did not affect in vitro reductase activity. Cleavage may be important in the modulation of the amino terminal-associated activity allowing it to interact with and modulate the activities of other proteins. In contrast to previous observations (Chung et al., 1989), transphosphorylation of histones by the HSV-2 R1 expressed in E.coli was not observed. That HSV-2 or HSV-1 R1 do not transphosphorylate can not be ruled out as the substrate may be very specific, protein kinases can have very specific substrates, it is also possible that a co-factor is required to promote transphosphorylation. The unique amino terminal region of HSV-1 R1, like that of HSV-2 R1, contains the intrinsic autophosphorylating activity. Mutations were made in HSV-1 R1 to localise and identify regions of R1 important in its autophosphorylating activity. The autophosphorylating activity was shown to reside within the first 257 amino acids of HSV-1, at least 70 amino acids of the unique region of R1 can be deleted without affecting the ability of this region to autophosphorylate. Mutation of the amino terminal region of R1 has identified regions which when mutated by the insertion of a linker either increase or decrease the autophosphorylating activity. An increased autophosphorylating activity was observed with two independent insertion mutants located within a similar region. This region may function normally to down-regulate the autophosphorylating activity possibly by masking sites of catalytic importance or sites of autophosphoryation, a conformational change may normally be required to overcome this inhibitory region. (Abstract shortened by ProQuest.).

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Virology
Date of Award: 1994
Depositing User: Enlighten Team
Unique ID: glathesis:1994-76390
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 19 Nov 2019 14:46
Last Modified: 19 Nov 2019 14:46

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