A Structure鈥揂ctivity Analysis for Probing the Mechanism of Processive Double-Stranded DNA Digestion by 位 Exonuclease Trimers

详细信息    查看全文

• 作者：Xinlei Pan ; Christopher E. Smith ; Jinjin Zhang ; Kimberly A. McCabe ; Jun Fu ; Charles E. Bell
• 刊名：Biochemistry
• 出版年：2015
• 出版时间：October 6, 2015
• 年：2015
• 卷：54
• 期：39
• 页码：6139-6148
• 全文大小：417K
• ISSN：1520-4995

文摘

位 exonuclease (位exo) is an ATP-independent 5鈥?to-3鈥?exonuclease that binds to double-stranded DNA (dsDNA) ends and processively digests the 5鈥?strand into mononucleotides. The crystal structure of 位exo 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鈥?strand is digested at one of the three active sites, and the 3鈥?strand passes through the narrow end of the channel to emerge out the back. This model was largely confirmed by the structure of the 位exo鈥揇NA complex, which further revealed that the enzyme unwinds the DNA by 2 bp prior to cleavage, to thread the 5鈥?end of the DNA into the active site. On the basis of this structure, an 鈥渆lectrostatic ratchet鈥?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鈥?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 位exo 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.