Ferrate Oxidation of Murine Leukemia Virus Reverse Transcriptase

Identification of the Template-Primer Binding Domain

Gurucharan Reddy, Venkata B. Nanduri, Amaresh Basu, Mukund Modak

Research output: Contribution to journalArticle

8 Citations (Scopus)

Abstract

Treatment of murine leukemia virus reverse transcriptase (MuLV RT) with potassium ferrate, an oxidizing agent known to oxidize amino acids involved in phosphate binding domains of proteins, results in the irreversible inactivation of both the DNA polymerase and the RNase H activities. Significant protection from ferrate-mediated inactivation is observed in the presence of template-primer but not in the presence of substrate deoxynucleoside triphosphates. Furthermore, ferrate-treated enzyme loses template-primer binding activity as judged by UV-mediated cross-linking of radiolabeled DNA. Comparative tryptic peptide mapping by reverse-phase HPLC of native and ferrate-oxidized enzyme indicated the presence of two new peptides eluting at 38 and 57 min and a significant loss of a peptide eluting at 74 min. Purification, amino acid composition, and sequencing of these affected peptides revealed that they correspond to amino acid residues 285-295, 630-640, and 586-599, respectively, in the primary amino acid sequence of MuLV RT. These results indicate that the domains constituted by the above peptides are important for the template-primer binding function in MuLV RT. Peptide I is located in the polymerase domain whereas peptides II and III are located in the RNase H domain. Amino acid sequence analysis of peptides I and II suggested Lys-285 and Cys-635 as the probable sites of ferrate action.

Original languageEnglish (US)
Pages (from-to)8195-8201
Number of pages7
JournalBiochemistry
Volume30
Issue number33
DOIs
StatePublished - Aug 1 1991

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Murine Leukemia Viruses
RNA-Directed DNA Polymerase
Viruses
Oxidation
Peptides
Ribonuclease H
Amino Acids
Protein Sequence Analysis
Phosphate-Binding Proteins
Peptide Mapping
DNA-Directed DNA Polymerase
Enzymes
Oxidants
ferrate ion
Amino Acid Sequence
High Pressure Liquid Chromatography
Purification
DNA
Phosphates
Substrates

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Reddy, Gurucharan ; Nanduri, Venkata B. ; Basu, Amaresh ; Modak, Mukund. / Ferrate Oxidation of Murine Leukemia Virus Reverse Transcriptase : Identification of the Template-Primer Binding Domain. In: Biochemistry. 1991 ; Vol. 30, No. 33. pp. 8195-8201.
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Ferrate Oxidation of Murine Leukemia Virus Reverse Transcriptase : Identification of the Template-Primer Binding Domain. / Reddy, Gurucharan; Nanduri, Venkata B.; Basu, Amaresh; Modak, Mukund.

In: Biochemistry, Vol. 30, No. 33, 01.08.1991, p. 8195-8201.

Research output: Contribution to journalArticle

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