Deoxynucleotide polymerization by HIV-1 reverse transcriptase is terminated by site-specific styrene oxide adducts after translesion synthesis

Gary J. Latham, Robert (Stephen) Lloyd

Research output: Contribution to journalArticle

36 Citations (Scopus)

Abstract

In an effort to integrate an understanding of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) structure with its function, the action of HIV-1 RT was examined in vitro on DNA templates modified with a model bulky DNA adduct. Styrene oxide was site-specifically and stereospecifically coupled to the N6 position of adenine to form six different adducted templates. Primer extension assays were conducted under conditions defining both single and multiple encounters between the polymerase and the damaged template-primer. The extent of polymerization observed for each adduct was found to depend on both the chirality of the damage and the lesion sequence context. When HIV-1 RT polymerization was limited by single encounters with damaged DNA, the SO lesions were readily bypassed as evidenced by minimal pausing at the adducted base. However, RT replication of all SO-modified templates was significantly terminated 3-5 nucleotides after translesion synthesis but before reaching the end of the template. These truncated products could be readily extended when additional encounters between enzyme and template-primer were permitted. A model is presented to explain these results in the context of HIV-1 RT structure during DNA replication.

Original languageEnglish (US)
Pages (from-to)28527-28530
Number of pages4
JournalJournal of Biological Chemistry
Volume269
Issue number46
StatePublished - Nov 18 1994
Externally publishedYes

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styrene oxide
Polymerization
HIV-1
DNA
DNA Adducts
Chirality
RNA-Directed DNA Polymerase
Adenine
DNA Replication
Assays
Nucleotides
Human immunodeficiency virus 1 reverse transcriptase
Enzymes

ASJC Scopus subject areas

  • Biochemistry

Cite this

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title = "Deoxynucleotide polymerization by HIV-1 reverse transcriptase is terminated by site-specific styrene oxide adducts after translesion synthesis",
abstract = "In an effort to integrate an understanding of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) structure with its function, the action of HIV-1 RT was examined in vitro on DNA templates modified with a model bulky DNA adduct. Styrene oxide was site-specifically and stereospecifically coupled to the N6 position of adenine to form six different adducted templates. Primer extension assays were conducted under conditions defining both single and multiple encounters between the polymerase and the damaged template-primer. The extent of polymerization observed for each adduct was found to depend on both the chirality of the damage and the lesion sequence context. When HIV-1 RT polymerization was limited by single encounters with damaged DNA, the SO lesions were readily bypassed as evidenced by minimal pausing at the adducted base. However, RT replication of all SO-modified templates was significantly terminated 3-5 nucleotides after translesion synthesis but before reaching the end of the template. These truncated products could be readily extended when additional encounters between enzyme and template-primer were permitted. A model is presented to explain these results in the context of HIV-1 RT structure during DNA replication.",
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N2 - In an effort to integrate an understanding of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) structure with its function, the action of HIV-1 RT was examined in vitro on DNA templates modified with a model bulky DNA adduct. Styrene oxide was site-specifically and stereospecifically coupled to the N6 position of adenine to form six different adducted templates. Primer extension assays were conducted under conditions defining both single and multiple encounters between the polymerase and the damaged template-primer. The extent of polymerization observed for each adduct was found to depend on both the chirality of the damage and the lesion sequence context. When HIV-1 RT polymerization was limited by single encounters with damaged DNA, the SO lesions were readily bypassed as evidenced by minimal pausing at the adducted base. However, RT replication of all SO-modified templates was significantly terminated 3-5 nucleotides after translesion synthesis but before reaching the end of the template. These truncated products could be readily extended when additional encounters between enzyme and template-primer were permitted. A model is presented to explain these results in the context of HIV-1 RT structure during DNA replication.

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