Fidelity of two retroviral reverse transcriptases during DNA-dependent DNA synthesis in vitro.

Abstract:

We determined the fidelity of avian myeloblastosis virus and Moloney murine leukemia virus reverse transcriptases (RTs) during DNA synthesis in vitro using the M13mp2 lacZ alpha gene as a mutational target. Both RTs commit an error approximately once for every 30,000 nucleotides polymerized. DNA sequence analysis of mutants generated in a forward mutation assay capable of detecting many types of errors demonstrated that avian myeloblastosis virus RT produced a variety of different mutations. The majority (58%) were single-base substitutions; all of which resulted from the misincorporation of either dAMP or dGMP. Minus-one frameshifts were also common, composing about 30% of the mutations. In addition to single-base events, eight mutants contained sequence changes involving from 2 to 59 bases. The frequency of these mutants suggests that, at least during DNA synthesis in vitro, RTs also commit errors by mechanisms other than classical base miscoding and misalignment. We examined the ability of RTs to synthesize DNA from a mismatched primer terminus at a sequence where the mismatched base was complementary to the next base in the template. Unlike cellular DNA polymerases which polymerize from the mismatched template-primer, RTs preferred to polymerize from a rearranged template-primer containing a matched terminal base pair and an unpaired base in the template strand. The unusual preference for this substrate suggests that the interactions between RTs and the template-primer are different from those of cellular DNA polymerases. The overall error rate of RT in vitro is sufficient to account for the estimated mutation rate of these viruses.

Polymerases:

Topics:

Reverse Transcriptase, Historical Protein Properties (MW, pI, ...), Exonuclease Activity, Fidelity, RNase H Activity, Accessory Proteins/Complexes

Status:

new topics/pols set partial results complete validated

Results:

Polymerase Reference Property Result Context
AMV Roberts JD1989 Reverse Transcriptase Activity Yes
AMV Roberts JD1989 Molecular Weight 158kD
AMV Roberts JD1989 3-5' Exonuclease (proofreading) No
AMV Roberts JD1989 Frameshift Error Rate 1.15E-05 errors/bp Technique: M13mp2 opal codon reversion assay
AMV Roberts JD1989 Nucleotide Substitution Rate 2.7E-05 errors/bp Technique: M13mp2 opal codon reversion assay
AMV Roberts JD1989 RNase H Yes
AMV Roberts JD1989 Other accessory protein(s) pp32pol phosphoprotein endonuclease
MMuLV RT Roberts JD1989 Reverse Transcriptase Activity Yes
MMuLV RT Roberts JD1989 Molecular Weight 84 Dalton
MMuLV RT Roberts JD1989 3-5' Exonuclease (proofreading) No
MMuLV RT Roberts JD1989 RNase H Yes

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