A Structure–Activity Analysis for Probing the Mechanism of Processive Double-Stranded DNA Digestion by λ Exonuclease Trimers

• Published in: Biochemistry (Easton) Vol. 54; no. 39; pp. 6139 - 6148
• Main Authors: Pan, Xinlei, Smith, Christopher E, Zhang, Jinjin, McCabe, Kimberly A, Fu, Jun, Bell, Charles E
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 06.10.2015
• Subjects: DNA - chemistry; Exodeoxyribonucleases - chemistry; Models, Chemical; Protein Multimerization; Protein Structure, Quaternary
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/acs.biochem.5b00707

λ exonuclease (λexo) is an ATP-independent 5′-to-3′ exonuclease that binds to double-stranded DNA (dsDNA) ends and processively digests the 5′-strand into mononucleotides. The crystal structure of λexo revealed that the enzyme forms a ring-shaped homotrimer with a central funnel-shaped channel for tracking along the DNA. On the basis of this structure, it was proposed that dsDNA enters the open end of the channel, the 5′-strand is digested at one of the three active sites, and the 3′-strand passes through the narrow end of the channel to emerge out the back. This model was largely confirmed by the structure of the λexo–DNA complex, which further revealed that the enzyme unwinds the DNA by 2 bp prior to cleavage, to thread the 5′-end of the DNA into the active site. On the basis of this structure, an “electrostatic ratchet” model was proposed, in which the enzyme uses a hydrophobic wedge to insert into the base pairs to unwind the DNA, a two-metal mechanism for nucleotide hydrolysis, a positively charged pocket to bind to the terminal 5′-phosphate generated after each round of cleavage, and an arginine residue (Arg-45) to bind to the minor groove of the downstream end of the DNA. To test this model, in this study we have determined the effects of 11 structure-based mutations in λexo on DNA binding and exonuclease activities in vitro, and on DNA recombination in vivo. The results are largely consistent with the model for the mechanism that was proposed on the basis of the structure and provide new insights into the roles of particular residues of the protein in promoting the reaction. In particular, a key role for Arg-45 in DNA binding is revealed.