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Journal of Bacteriology, September 2004, p. 5699-5707, Vol. 186, No. 17
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.17.5699-5707.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Transposon-Mediated Linker Insertion Scanning Mutagenesis of the Escherichia coli McrA Endonuclease

Brian P. Anton and Elisabeth A. Raleigh*

New England Biolabs, Beverly, Massachusetts

Received 30 March 2004/ Accepted 27 May 2004

McrA is one of three functions that restrict modified foreign DNA in Escherichia coli K-12, affecting both methylated and hydroxymethylated substrates. We present here the first systematic analysis of the functional organization of McrA by using the GPS-LS insertion scanning system. We collected in-frame insertions of five amino acids at 46 independent locations and C-terminal truncations at 20 independent locations in the McrA protein. Each mutant was assayed for in vivo restriction of both methylated and hydroxymethylated bacteriophage (M.HpaII-modified {lambda} and T4gt, respectively) and for induction of the E. coli SOS response in the presence of M.HpaII methylation, indicative of DNA damage. Our findings suggest the presence of an N-terminal DNA-binding domain and a C-terminal catalytic nuclease domain connected by a linker region largely tolerant of amino acid insertions. DNA damage inflicted by a functional C-terminal domain is required for restriction of phage T4gt. Disruption of the N-terminal domain abolishes restriction of both substrates. Surprisingly, truncation mutations that spare the N-terminal domain do not mediate DNA damage, as measured by SOS induction, but nevertheless partially restrict M.HpaII-modified {lambda} in vivo. We suggest a common explanation for this "restriction without damage" and a similar observation seen in vivo with McrB, a component of another of the modified-DNA restriction functions. Briefly, we propose that unproductive site-specific binding of the protein to a vulnerable position in the {lambda} genome disrupts the phage development program at an early stage. We also identified a single mutant, carrying an insertion in the N-terminal domain, which could fully restrict {lambda} but did not restrict T4gt at all. This mutant may have a selective impairment in substrate recognition, distinguishing methylated from hydroxymethylated substrates. The study shows that the technically easy insertion scanning method can provide a rich source of functional information when coupled with effective phenotype tests.

* Corresponding author. Mailing address: New England Biolabs, 32 Tozer Rd., Beverly, MA 01915. Phone: (978) 927-5054. Fax: (978) 921-1350. E-mail: raleigh{at}neb.com.

Journal of Bacteriology, September 2004, p. 5699-5707, Vol. 186, No. 17
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.17.5699-5707.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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