Mutant Poisoning Demonstrates a Nonsequential Mechanism for Digestion of Double-Stranded DNA by 位 Exonuclease Trimers

详细信息    查看全文

• 作者：Xinlei Pan ; Jing Yan ; Aalapi Patel ; Vicki H. Wysocki ; Charles E. Bell
• 刊名：Biochemistry
• 出版年：2015
• 出版时间：January 27, 2015
• 年：2015
• 卷：54
• 期：3
• 页码：942-951
• 全文大小：400K
• ISSN：1520-4995

文摘

位 Exonuclease (位exo) is a highly processive 5鈥?3鈥?exonuclease that binds double-stranded DNA (dsDNA) ends and digests the 5鈥?strand into mononucleotides. The enzyme forms a toroidal homotrimer with a central tapered channel for tracking along the DNA. During catalysis, dsDNA enters the open end of the channel, and the 5鈥?strand is digested at one of the three active sites. It is currently not known if 位exo uses a sequential mechanism, in which the DNA moves from one active site to the next around the trimer for each round of catalysis or a nonsequential mechanism, in which the DNA locks onto a single active site for multiple rounds. To understand how 位exo uses its three active sites, we used a mutant poisoning approach, in which a 6xHis-tagged K131A inactive mutant of 位exo was mixed with untagged wild type (WT) to form hybrid trimers. Nickel-spin pull-down analysis confirmed complete subunit exchange after 1 h at 37 掳C. Exonuclease assays revealed an approximately linear decrease in activity with increasing fraction of mutant, as expected for a nonsequential mechanism. By fitting the observed rates of digestion to a simple mathematical model, the individual rates of the two hybrid species of trimer were determined. This analysis showed that trimers containing only one or two WT subunits contribute significantly to the observed activity, in further agreement with a nonsequential mechanism. Finally, purification of hybrid trimer mixtures by Ni-spin chromatography, to remove the contribution from fully WT trimers, also resulted in significant levels of activity, again consistent with a nonsequential mechanism.