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Crystal structure of xenotropic murine leukaemia virus-related virus (XMRV) ribonuclease H.

Kim JH, Kang S, Jung SK, Yu KR, Chung SJ, Chung BH, Erikson RL, Kim BY, Kim SJ - Biosci. Rep. (2012)

Bottom Line: RNase H (retroviral ribonuclease H) cleaves the phosphate backbone of the RNA template within an RNA/DNA hybrid to complete the synthesis of double-stranded viral DNA.In the present study we have determined the complete structure of the RNase H domain from XMRV (xenotropic murine leukaemia virus-related virus) RT (reverse transcriptase).The basic protrusion motif of the XMRV RNase H domain is folded as a short helix and an adjacent highly bent loop.

View Article: PubMed Central - PubMed

Affiliation: Medical Proteomics Research Center, Korea Research Institute of Bioscience and Biotechnology, Yuseong-Gu, Daejeon, Republic of Korea.

ABSTRACT
RNase H (retroviral ribonuclease H) cleaves the phosphate backbone of the RNA template within an RNA/DNA hybrid to complete the synthesis of double-stranded viral DNA. In the present study we have determined the complete structure of the RNase H domain from XMRV (xenotropic murine leukaemia virus-related virus) RT (reverse transcriptase). The basic protrusion motif of the XMRV RNase H domain is folded as a short helix and an adjacent highly bent loop. Structural superposition and subsequent mutagenesis experiments suggest that the basic protrusion motif plays a role in direct binding to the major groove in RNA/DNA hybrid, as well as in establishing the co-ordination among modules in RT necessary for proper function.

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Categorization and location of mutationsResidues at which mutations were introduced are shown as sticks on the ribbon diagram. The mutated residues are coloured differently according to the roles of mutations (His-containing loop, cyan; hydrophilic contact, green; hydrophobic contact, yellow).
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Figure 4: Categorization and location of mutationsResidues at which mutations were introduced are shown as sticks on the ribbon diagram. The mutated residues are coloured differently according to the roles of mutations (His-containing loop, cyan; hydrophilic contact, green; hydrophobic contact, yellow).

Mentions: In order to confirm the functional importance of residues suggested to interact with RNA/DNA hybrid based on the crystal structure, we individually mutated the identified residues and tested catalytic activity. We created three groups of XMRV RNase H mutants based on the structural information on XMRV RNase H (Figure 4). These groups are designed to show how three motifs, the His-containing loop, hydrophilic contacts in the basic protrusion and hydrophobic contacts in the basic protrusion, affect RT catalytic activity (Figure 4, Table 2). All mutations except for Val593 involved alanine substitution to exclude the effects of side chain. Val593 was replaced with the charged residue aspartate to test whether hydrophobic contact is important. Initial studies showed that the catalytic activity of the RNase H domain alone is too low to allow the determination of kinetic parameters. Therefore we inserted these mutations into full-length XMRV RT enzymes. All mutant RT enzymes were successfully overexpressed in E. coli and their purities were determined to be greater than 90% by SDS/PAGE (results not shown). Catalytic activity was determined using fluorescein-labelled RNA/DNA duplex as a substrate [30]. Kinetic constants of wild-type and mutant enzymes are summarized in Table 2.


Crystal structure of xenotropic murine leukaemia virus-related virus (XMRV) ribonuclease H.

Kim JH, Kang S, Jung SK, Yu KR, Chung SJ, Chung BH, Erikson RL, Kim BY, Kim SJ - Biosci. Rep. (2012)

Categorization and location of mutationsResidues at which mutations were introduced are shown as sticks on the ribbon diagram. The mutated residues are coloured differently according to the roles of mutations (His-containing loop, cyan; hydrophilic contact, green; hydrophobic contact, yellow).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3475449&req=5

Figure 4: Categorization and location of mutationsResidues at which mutations were introduced are shown as sticks on the ribbon diagram. The mutated residues are coloured differently according to the roles of mutations (His-containing loop, cyan; hydrophilic contact, green; hydrophobic contact, yellow).
Mentions: In order to confirm the functional importance of residues suggested to interact with RNA/DNA hybrid based on the crystal structure, we individually mutated the identified residues and tested catalytic activity. We created three groups of XMRV RNase H mutants based on the structural information on XMRV RNase H (Figure 4). These groups are designed to show how three motifs, the His-containing loop, hydrophilic contacts in the basic protrusion and hydrophobic contacts in the basic protrusion, affect RT catalytic activity (Figure 4, Table 2). All mutations except for Val593 involved alanine substitution to exclude the effects of side chain. Val593 was replaced with the charged residue aspartate to test whether hydrophobic contact is important. Initial studies showed that the catalytic activity of the RNase H domain alone is too low to allow the determination of kinetic parameters. Therefore we inserted these mutations into full-length XMRV RT enzymes. All mutant RT enzymes were successfully overexpressed in E. coli and their purities were determined to be greater than 90% by SDS/PAGE (results not shown). Catalytic activity was determined using fluorescein-labelled RNA/DNA duplex as a substrate [30]. Kinetic constants of wild-type and mutant enzymes are summarized in Table 2.

Bottom Line: RNase H (retroviral ribonuclease H) cleaves the phosphate backbone of the RNA template within an RNA/DNA hybrid to complete the synthesis of double-stranded viral DNA.In the present study we have determined the complete structure of the RNase H domain from XMRV (xenotropic murine leukaemia virus-related virus) RT (reverse transcriptase).The basic protrusion motif of the XMRV RNase H domain is folded as a short helix and an adjacent highly bent loop.

View Article: PubMed Central - PubMed

Affiliation: Medical Proteomics Research Center, Korea Research Institute of Bioscience and Biotechnology, Yuseong-Gu, Daejeon, Republic of Korea.

ABSTRACT
RNase H (retroviral ribonuclease H) cleaves the phosphate backbone of the RNA template within an RNA/DNA hybrid to complete the synthesis of double-stranded viral DNA. In the present study we have determined the complete structure of the RNase H domain from XMRV (xenotropic murine leukaemia virus-related virus) RT (reverse transcriptase). The basic protrusion motif of the XMRV RNase H domain is folded as a short helix and an adjacent highly bent loop. Structural superposition and subsequent mutagenesis experiments suggest that the basic protrusion motif plays a role in direct binding to the major groove in RNA/DNA hybrid, as well as in establishing the co-ordination among modules in RT necessary for proper function.

Show MeSH
Related in: MedlinePlus