Comparison of DNA polymerase activities between recombinant feline immunodeficiency and leukemia virus reverse transcriptases.

Abstract:

In this study, we present enzymatic differences found between recombinant RTs derived from feline leukemia virus and feline immunodeficiency virus. Firstly, FIV RT showed low steady state K(m) values for dNTPs compared to FeLV RT. Consistent with this, FIV RT synthesized DNA more efficiently than FeLV RT at low dNTP concentrations. We observed similar concentration-dependent activity differences between other lentiviral (HIV-1 and SIV) and non-lentiviral (MuLV and AMV) RTs. Second, FeLV RT showed less efficient misincorporation with biased dNTP pools and mismatch primer extension capabilities, compared to FIV RT. In M13mp2 lacZalpha forward mutation assays, FeLV RT displayed approximately 11-fold higher fidelity than FIV RT. Finally, FeLV RT was less sensitive to 3TCTP and ddATP than FIV RT. This study represents the comprehensive enzymatic characterization of RTs from a lentivirus and a non-lentivirus retrovirus from the same host species. The data presented here support enzymatic divergences seen among retroviral RTs.

Polymerases:

Topics:

Historical Protein Properties (MW, pI, ...), Kinetic Parameters, Fidelity, RNase H Activity, Nucleotide Incorporation, Reverse Transcriptase, Source / Purification

Status:

new topics/pols set partial results complete validated

Results:

Polymerase Reference Property Result Context
HIV RT Operario DJ2005 Molecular Weight 66kD
HIV RT Operario DJ2005 Overall Error Rate 0.0005 errors/bp
MMuLV RT Operario DJ2005 Molecular Weight 71kD
FIV RT Operario DJ2005 Reverse Transcriptase Activity Yes
FIV RT Operario DJ2005 Cloned or native Cloned in E. coli
FIV RT Operario DJ2005 Tagged Yes
FIV RT Operario DJ2005 Tag Name His
FIV RT Operario DJ2005 Overall Error Rate 0.00942 errors/bp Technique: M13mp2 forward mutation assay
FIV RT Operario DJ2005 Full length or truncated Full length
FIV RT Operario DJ2005 RNase H Yes
FIV RT Operario DJ2005 KM 0.06uM Reaction: Nucleotide incorporation; Substrate: dGTP
FIV RT Operario DJ2005 KM 0.081uM Reaction: Nucleotide incorporation; Substrate: dATP
FIV RT Operario DJ2005 KM 0.051uM Reaction: Nucleotide incorporation; Substrate: dTTP
FIV RT Operario DJ2005 KM 0.028uM Reaction: Nucleotide incorporation; Substrate: dCTP
FIV RT Operario DJ2005 kcat 0.51 /second Reaction: Nucleotide incorporation; Substrate: dGTP
FIV RT Operario DJ2005 kcat 0.21 /second Reaction: Nucleotide incorporation; Substrate: dATP
FIV RT Operario DJ2005 kcat 0.28 /second Reaction: Nucleotide incorporation; Substrate: dTTP
FIV RT Operario DJ2005 kcat 0.22 /second Reaction: Nucleotide incorporation; Substrate: dCTP
FIV RT Operario DJ2005 Gap Filling Yes
FeLV RT Operario DJ2005 Reverse Transcriptase Activity Yes
FeLV RT Operario DJ2005 Cloned or native Cloned in E. coli
FeLV RT Operario DJ2005 Tagged Yes
FeLV RT Operario DJ2005 Tag Name His
FeLV RT Operario DJ2005 Overall Error Rate 0.00088 errors/bp Technique: M13mp2 forward mutation assay
FeLV RT Operario DJ2005 Full length or truncated Full length
FeLV RT Operario DJ2005 RNase H Yes
FeLV RT Operario DJ2005 KM 0.936uM Reaction: Nucleotide incorporation; Substrate: dGTP
FeLV RT Operario DJ2005 KM 4.674uM Reaction: Nucleotide incorporation; Substrate: dATP
FeLV RT Operario DJ2005 KM 2.546uM Reaction: Nucleotide incorporation; Substrate: dTTP
FeLV RT Operario DJ2005 KM 1.481n/a Reaction: Nucleotide incorporation; Substrate: dCTP
FeLV RT Operario DJ2005 kcat 0.56 /second Reaction: Nucleotide incorporation; Substrate: dGTP
FeLV RT Operario DJ2005 kcat 0.18 /second Reaction: Nucleotide incorporation; Substrate: dATP
FeLV RT Operario DJ2005 kcat 0.17 /second Reaction: Nucleotide incorporation; Substrate: dTTP
FeLV RT Operario DJ2005 kcat 0.15 /second Reaction: Nucleotide incorporation; Substrate: dCTP
FeLV RT Operario DJ2005 Gap Filling Yes

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