A Single Nuclease Active Site of the Escherichia coli RecBCD Enzyme Catalyzes Single-stranded DNA Degradation in Both Directions

Jingdi Wang,Ruiwu Chen,Douglas A Julin

doi:10.1074/jbc.275.1.507

Jingdi Wang, Ruiwu Chen + Show 1 more

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https://doi.org/10.1074/jbc.275.1.507

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Abstract

The RecBCD enzyme of Escherichia coli is an ATP-dependent DNA exonuclease and a helicase. Its exonuclease activity is subject to regulation by an octameric nucleotide sequence called chi. In this study, site-directed mutations were made in the carboxyl-terminal nuclease domain of the RecB subunit, and their effects on RecBCD's enzymatic activities were investigated. Mutation of two amino acid residues, Asp(1067) and Lys(1082), abolished nuclease activity on both single- and double-stranded DNA. Together with Asp(1080), these residues compose a motif that is similar to one shown to form the active site of several restriction endonucleases. The nuclease reactions catalyzed by the RecBCD enzyme should therefore follow the same mechanism as these restriction endonucleases. Furthermore, the mutant enzymes were unable to produce chi-specific fragments that are thought to result from the 3'-5' and 5'-3' single-stranded exonuclease activities of the enzyme during its reaction with chi-containing double-stranded DNA. The results show that the nuclease active site in the RecB C-terminal 30-kDa domain is the universal nuclease active site of RecBCD that is responsible for DNA degradation in both directions during the reaction with double-stranded DNA. A novel explanation for the observed nuclease polarity switch and RecBCD-DNA interaction is offered.

Highlights

The RecBCD enzyme of Escherichia coli is a trimeric, multifunctional enzyme composed of the RecB, RecC, and RecD subunits, whose calculated molecular masses are 134, 129, and 67 kDa, respectively (4 – 6)
Analyses on the E. coli chromosome have revealed that the density of ␹ is higher in the proximity of the origin of replication, oriC, and these ␹ sites are favorably oriented to protect oriC from degradative RecBCDs approaching from both sides [17]
The Nuclease Active Site in RecBCD: Comparison with Other Nucleases—Three of the amino acid residues of RecB altered in this study, Asp1067, Lys1082, and Tyr1114, along with Asp1080, previously changed to alanine by Yu et al [25], are highly conserved among RecB homologs (Fig. 1) and a number of other proteins that have been called the “RecB nuclease domain” family [34]

Summary

Introduction

The RecBCD enzyme of Escherichia coli (exonuclease V; reviewed in Refs. 1–3) is a trimeric, multifunctional enzyme composed of the RecB, RecC, and RecD subunits, whose calculated molecular masses are 134, 129, and 67 kDa, respectively (4 – 6). RecBCD helps E. coli to recover from double strand breaks in its chromosome during genomic replication [9] This function of the enzyme is believed. The enzyme can cleave the strand containing the complement of the ␹ sequence and 5Ј-terminated at the entry site, in a ␹-specific manner [22], and cleavage of this strand was found to increase after RecBCD passes the ␹ sequence [23]. These observations led to the proposal that ␹, in addition to down-regulating RecBCD’s 3Ј-to-5Ј ssDNA exonuclease activity, up-regulates its 5Ј-to-3Ј ssDNA exonuclease activity on the other strand. Recent work has shown that the RecB subunit, in addition to its DNA helicase activity [24], has a critical role in the intricate

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