The K65R mutation confers increased DNA polymerase processivity to HIV-1 reverse transcriptase.

Abstract:

The K65R mutation in HIV-1 reverse transcriptase (RT) is associated with viral cross-resistance to 2',3'-dideoxyinosine, 2',3'-dideoxycytidine, and 2',3'-dideoxy-3'-thiacytidine. We have found that in vitro DNA synthesis by K65R RT is significantly more processive than that of wild type (wt) RT. Depending on the template/primer (T/P) used, the total incorporation of nucleotides under single processive cycle conditions was 20-50% higher with K65R RT than with wt RT. With heteropolymeric T/P, the total incorporation of dNMP by K65R and wt RT was similar under continuous DNA synthesis reaction conditions. However, under single processive cycle conditions, the rate of full-length polymerization product synthesis by K65R RT was about 2-fold higher than that by wt RT. We also found a decreased rate of T/P dissociation during K65R RT DNA synthesis, which is consistent with the increased processivity of the enzyme. We postulate that the increased processivity of the K65R RT may be a compensatory response to the decreased affinity of this mutant for certain dNTP substrates, allowing normal viral replication kinetics.

Polymerases:

Topics:

Other Enzymatic Activities, Reverse Transcriptase, Source / Purification

Status:

new topics/pols set partial results complete validated

Results:

Polymerase Reference Property Result Context
HIV RT Arion D1996 Reverse Transcriptase Activity Yes
HIV RT Arion D1996 Cloned or native Cloned in E. coli
HIV RT Arion D1996 Full length or truncated Full length
HIV RT Arion D1996 Extension from RNA primer Yes
HIV RT K65R Arion D1996 Reverse Transcriptase Activity Yes
HIV RT K65R Arion D1996 Cloned or native Cloned in E. coli
HIV RT K65R Arion D1996 Full length or truncated Full length
HIV RT K65R Arion D1996 Extension from RNA primer Yes

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