Evidence for an imino intermediate in the dna polymerase beta deoxyribose phosphate excision reaction

C. Piersen, R. Prasad, S. Wilson, Robert (Stephen) Lloyd

Research output: Contribution to journalArticle

Abstract

A recent study demonstrated that rat DNA polymerase beta (beta-pol) releases 5′-deoxyribose phosphate (dRP) termini from preincised apurinic/apyrimidinic DNA, a substrate generated during some types of base excision repair. This catalytic activity resides within the amino-terminal, 8-kDa domain of beta-pol and occurs via beta-elimination (Matsumoto, Y., and Kim, K. (1995) Science 269, 699-702). The last finding suggested to us that the dRP excision reaction might proceed via an imine intermediate. In order to test this hypothesis, we attempted to trap beta-pol on preincised apurinic/apyrimidinic DNA using NaBH4 as the reducing agent. Both 8-kDa domain- and intact beta-pol-DNA complexes were detected and identified by autoradiography coupled with immunoblotting. Helix-2 and its adjacent loops do not appear to be involved directly in catalysis; the corresponding 32-amino acid peptide (residues 27-58) could not be covalently trapped, and neither the peptide itself nor an antipeptide antibody could inhibit trapping of the 8-kDa domain (residues 1-87). Thus, the active site residue responsible for dRP release by beta-pol is proposed to be a primary amine, presumably one of the 9 lysines at the amino- or carboxyl-terminal end of the 8-kDa domain.

Original languageEnglish (US)
JournalFASEB Journal
Volume10
Issue number6
StatePublished - 1996
Externally publishedYes

Fingerprint

Deoxyribose
Phosphates
phosphates
catalytic activity
DNA
peptides
DNA Polymerase beta
primary amines
Peptides
Imines
reducing agents
Reducing Agents
autoradiography
DNA-directed DNA polymerase
DNA repair
immunoblotting
Autoradiography
Catalysis
Immunoblotting
active sites

ASJC Scopus subject areas

  • Agricultural and Biological Sciences (miscellaneous)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Biochemistry
  • Cell Biology

Cite this

Evidence for an imino intermediate in the dna polymerase beta deoxyribose phosphate excision reaction. / Piersen, C.; Prasad, R.; Wilson, S.; Lloyd, Robert (Stephen).

In: FASEB Journal, Vol. 10, No. 6, 1996.

Research output: Contribution to journalArticle

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AU - Piersen, C.

AU - Prasad, R.

AU - Wilson, S.

AU - Lloyd, Robert (Stephen)

PY - 1996

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