Poster abstracts

Poster number 57 submitted by Xinlei Pan

A mutant poisoning approach to determine if λ Exonuclease trimers use a sequential or non-sequential mechanism for processive digestion of dsDNA substrates

Xinlei Pan (Department of Molecular and Cellullar Biochemistry, the Ohio State University), Charles E. Bell (Department of Molecular and Cellullar Biochemistry, the Ohio State University)

Abstract:
λ Exonuclease is a highly processive 5’-3’ exonuclease that binds double-stranded DNA ends and digests the 5’ end into mononucleotides. The digestion product, a 3’ single-stranded overhang DNA, can serve as the substrate for pairing proteins in homologous recombination. λ Exonuclease forms a homotrimeric ring with a tapered central channel for tracking along the DNA. During catalysis, dsDNA enters the wider side of the channel and the 5’ end is digested at one of the three active centers. Then the resulting 3’ single-stranded overhang exits through the narrower side of the channel to emerge out the back of the trimer. Two mechanisms could be envisioned for enzymes that form oligomeric rings to use their multiple active sites: the sequential mechanism, where all active sites in the oligomer are engaged in catalysis sequentially; and the non-sequential mechanism, where the substrate DNA locks onto one active site for multiple rounds of catalysis. To understand how the λ Exonuclease trimer uses its three active sites, we used a “mutant poisoning” approach, where inactive subunits are mixed with active subunits to form hybrid trimers. In the sequential mechanism, one inactive subunit will cause the trimer to lose all activity; whereas in the non-sequential mechanism, the trimer remains active as long as there is at least one active subunit. A K131A mutant of λ Exonuclease, which is completely inactive for nucleotide hydrolysis, but maintains its DNA binding ability, was introduced. Nickel spin pull down assays, where K131A mutant with a Histidine tag was mixed with untagged wild-type λ Exonuclease and run on a nickel column, showed observable subunit exchange after 1h incubation at 37℃. The λ Exonuclease activity was measured by determining the rate of digestion of a linear pUC19 duplex DNA. The results showed that the λ Exonuclease hybrid trimers still remain active, suggesting that a non-sequential mechanism of active sites is used during λ Exonuclease catalysis.

References:
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Zhang J., McCabe K.A., and Bell C.E. (2011) Crystal structures of lambda exonuclease in complex with DNA suggest an electrostatic ratchet mechanism for processivity. Proc. Natl. Acad. Sci. 29: 11872-7.
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Keywords: Homologous Recombination, Lambda Exonuclease, mutant poisoning