Probing the Flexibility of the Catalytic Nucleophile in the Lyase Catalytic Pocket of Human DNA Polymerase β with Unnatural Lysine Analogues

Sasha M. Daskalova, Chandrabali Bhattacharya, Larisa Dedkova, Sidney Hecht

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

DNA polymerase β (Pol β) is a key enzyme in mammalian base excision repair (BER), contributing stepwise 5′-deoxyribose phosphate (dRP) lyase and "gap-filling" DNA polymerase activities. The lyase reaction is believed to occur via a β-elimination reaction following the formation of a Schiff base between the dRP group at the pre-incised apurinic/apyrimidinic site and the μ-amino group of Lys72. To probe the steric constraints on the formation and subsequent resolution of the putative Schiff base intermediate within the lyase catalytic pocket, Lys72 was replaced with each of several nonproteinogenic lysine analogues. The modified Pol β enzymes were produced by coupled in vitro transcription and translation from a modified DNA template containing a TAG codon at the position corresponding to Lys72. In the presence of a misacylated tRNACUA transcript, suppression of the UAG codon in the transcribed mRNA led to elaboration of full length Pol β having a lysine analogue at position 72. Replacement of the primary nucleophilic amine with a secondary amine in the form of N-methyllysine (4) affected mainly the stability of the Schiff base intermediate and resulted in relatively moderate inhibition of lyase activity and BER. Elongation of the side chain of the catalytic residue by one methylene group, achieved by introduction of homolysine (6) at position 72, apparently shifted the amino group to a position less favorable for Schiff base formation. Interestingly, this effect was attenuated when the side chain was elongated by replacing one side-chain methylene group with a bridging S atom (thialysine, 2). In comparison, replacement of lysine 72 with an analogue having a guanidine moiety in lieu of an μ-amino group (homoarginine, 5) or a sterically constrained secondary amine (piperidinylalanine, 3) led to almost complete suppression of dRP excision activity and the ability of Pol β to support BER. These results help to define the tolerance of Pol β to subtle local structural and functional alterations.

Original languageEnglish (US)
Pages (from-to)500-513
Number of pages14
JournalBiochemistry
Volume56
Issue number3
DOIs
StatePublished - Jan 24 2017

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Nucleophiles
Lyases
Schiff Bases
DNA-Directed DNA Polymerase
Lysine
DNA Repair
Deoxyribose
Amines
Repair
Terminator Codon
Homoarginine
Phosphates
Guanidine
Enzymes
Transcription
Elongation
Atoms
Messenger RNA
DNA

ASJC Scopus subject areas

  • Biochemistry

Cite this

Probing the Flexibility of the Catalytic Nucleophile in the Lyase Catalytic Pocket of Human DNA Polymerase β with Unnatural Lysine Analogues. / Daskalova, Sasha M.; Bhattacharya, Chandrabali; Dedkova, Larisa; Hecht, Sidney.

In: Biochemistry, Vol. 56, No. 3, 24.01.2017, p. 500-513.

Research output: Contribution to journalArticle

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abstract = "DNA polymerase β (Pol β) is a key enzyme in mammalian base excision repair (BER), contributing stepwise 5′-deoxyribose phosphate (dRP) lyase and {"}gap-filling{"} DNA polymerase activities. The lyase reaction is believed to occur via a β-elimination reaction following the formation of a Schiff base between the dRP group at the pre-incised apurinic/apyrimidinic site and the μ-amino group of Lys72. To probe the steric constraints on the formation and subsequent resolution of the putative Schiff base intermediate within the lyase catalytic pocket, Lys72 was replaced with each of several nonproteinogenic lysine analogues. The modified Pol β enzymes were produced by coupled in vitro transcription and translation from a modified DNA template containing a TAG codon at the position corresponding to Lys72. In the presence of a misacylated tRNACUA transcript, suppression of the UAG codon in the transcribed mRNA led to elaboration of full length Pol β having a lysine analogue at position 72. Replacement of the primary nucleophilic amine with a secondary amine in the form of N-methyllysine (4) affected mainly the stability of the Schiff base intermediate and resulted in relatively moderate inhibition of lyase activity and BER. Elongation of the side chain of the catalytic residue by one methylene group, achieved by introduction of homolysine (6) at position 72, apparently shifted the amino group to a position less favorable for Schiff base formation. Interestingly, this effect was attenuated when the side chain was elongated by replacing one side-chain methylene group with a bridging S atom (thialysine, 2). In comparison, replacement of lysine 72 with an analogue having a guanidine moiety in lieu of an μ-amino group (homoarginine, 5) or a sterically constrained secondary amine (piperidinylalanine, 3) led to almost complete suppression of dRP excision activity and the ability of Pol β to support BER. These results help to define the tolerance of Pol β to subtle local structural and functional alterations.",
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