Alteration of χ recognition by RecBCD reveals a regulated molecular latch and suggests a channel-bypass mechanism for biological control Liang Yanga,1, Naofumi Handaa,b,1, Bian Liua, Mark S. Dillinghamc, Dale B. Wigleyd, and Stephen C. Kowalczykowskia,2 aDepartment of Microbiology and Department of Molecular and Cellular Biology, University of California, Davis, CA 95616; bDepartment of Medical Genome Sciences, Graduate School of Frontier Sciences, University of Tokyo, Shirokanedai, Minato-ku, Tokyo 108-8639, Japan; cDNA-Protein Interactions Unit, School of Biochemistry, University of Bristol, Bristol BS8 1TD, United Kingdom; and dDivision of Structural Biology, Chester Beatty Laboratories, Institute of Cancer Research, London SW3 6JB, United Kingdom Contributed by Stephen C. Kowalczykowski, April 12, 2012 (sent for review February 21, 2012) The RecBCD enzyme is a complex heterotrimeric helicase/nuclease interaction with χ, RecBCD also briefly pauses and then resumes that initiates recombination at double-stranded DNA breaks. In translocation, but at approximately half the initial rate (15–17). Escherichia coli, its activities are regulated by the octameric recom- Because the helicase activity is retained (9), ssDNA is produced bination hotspot, χ (5′-GCTGGTGG), which is read as a single- with χ at its 3′-terminus, which is the optimal substrate for RecA- stranded DNA sequence while the enzyme is unwinding DNA at promoted invasion of dsDNA (14). Thus, interaction with χ over ∼1,000 bp/s. Previous studies implicated the RecC subunit as changes RecBCD from its “destructive” mode, digesting DNA as the “χ-scanning element” in this process. Site-directed mutagene- it unwinds, to a “recombinational” mode, preserving the χ-con- sis and phenotypic analyses identified residues in RecC responsible taining ssDNA and loading RecA onto this ssDNA (1, 2); these for χ recognition [Handa N, et al., (2012) Proc Natl Acad Sci USA, are two essential biological functions of the RecBCD–χ inter- 10.1073/pnas.1206076109]. The genetic analyses revealed two action (18, 19). classes of mutants. Here we use ensemble and single-molecule The crystal structure of RecBCD bound to a DNA hairpin criteria to biochemically establish that one class of mutants (type provided a great deal of information in understanding RecBCD 1) has lost the capacity to recognize χ (lost-recognition), whereas function, in particular with regard to the potential role of RecC χ the second class (type 2) has a lowered specificity for recognition in recognition (20). Strikingly, RecC displays the fold of a ca- (relaxed-specificity). The relaxed-specificity mutants still recognize nonical UvrD-like helicase, even though none of the character- canonical χ, but they have gained the capacity to precociously istic Superfamily 1 (SF1) helicase motifs are preserved. There fl recognize single-nucleotide variants of χ. Based on the RecBCD are three holes in RecC: a large one anked by two smaller ones. structure, these mutant classes define an α-helix responsible for The large hole serves as the interface between the RecB and χ RecC subunits. One of the small tunnels feeds the 5′-terminated recognition that is allosterically coupled to a structural latch. ′ When opened, we propose that the latch permits access to an ssDNA strand to RecD, and the other feeds the 3 -terminated strand from RecB, through RecC, into a nuclease domain im- alternative exit channel for the single-stranded DNA downstream “ ” ′ of χ, thereby avoiding degradation by the nuclease domain. These mediately behind the RecB motor domains. The 3 -strand fi tunnel in RecC is formed by the helicase-like domains, and the ndings provide a unique perspective into the mechanism by recC fi fi χ which recognition of a single-stranded DNA sequence switches * mutants, which alter the ef ciency and speci city of recognition, map to this region (21–23). Consequently, it was the translocating RecBCD from a destructive nuclease to a construc- proposed that this tunnel region functions to channel a correctly tive component of recombinational DNA repair. oriented χ sequence for recognition by RecC. In the companion article by Handa et al. (24), amino acids protein–DNA interactions | allosteric switch within the RecC channel were altered by site-directed muta- BIOCHEMISTRY genesis to identify the residues responsible for χ recognition. The n Escherichia coli, homologous recombination is initiated by genetic analyses discovered two types of mutants (Fig. S1). The Ithe RecBCD enzyme (1). RecBCD is a helicase/nuclease that first category, referred to as type 1, displayed phenotypic char- processes linear double-stranded DNA (dsDNA) resulting from acteristics consistent with a complete loss of the χ response; dsDNA breaks (2) to produce single-stranded DNA (ssDNA) these mutants included L64A, W70A, D133A, D136A, and onto which it loads the RecA protein (3). These RecA-ssDNA R186A, and recombination in these cells was not responsive to χ. filaments are essential to the formation of homologously paired In contrast, the second category, referred to as type 2, displayed joint molecules that are intermediates of recombinational DNA- phenotypic characteristics consistent with a promiscuous response break repair. to χ: these mutants included Q38A, T40A, L134A, Q137A, R142A, fi ∼ – RecBCD binds to dsDNA ends with high af nity (KM 0.1 1 and D705A. To understand the biochemical basis for these nM) (4, 5). It is a rapid and highly processive helicase, unwinding complex phenotypes in vivo, six mutant proteins of each type DNA at up to 1,000–1,500 bp/s, translocating about 30,000 bp were purified and their in vitro behavior was investigated. We before dissociating, and using ∼2 ATP molecules per base pair found that the type 1 mutants represent a group of RecBCD unwound (4–6). The RecBCD enzyme is driven by two motor enzymes that fail to recognize χ, and that the type 2 mutants subunits, RecB and RecD, with opposite translocation polarities, 3′ →5′ and 5′ →3′, respectively (7, 8). As it is unwinding, RecBCD ′ degrades the nascent ssDNA, preferentially on the 3 -teminated Author contributions: L.Y., N.H., M.S.D., D.B.W., and S.C.K. designed research; L.Y., N.H., strand relative to its DNA entry site (9). and B.L. performed research; L.Y., N.H., and B.L. contributed new reagents/analytic tools; RecBCD is regulated by the recombination hotspot, χ, the L.Y., N.H., B.L., M.S.D., D.B.W., and S.C.K. analyzed data; and L.Y., N.H., B.L., M.S.D., D.B.W., octameric DNA sequence (5′-GCTGGTGG-3′) (10), which is and S.C.K. wrote the paper. recognized from its 3′-side as ssDNA by the translocating en- The authors declare no conflict of interest. zyme (9, 11, 12). In response to χ recognition, the polarity of 1L.Y. and N.H. contributed equally to this work. RecBCD nucleolytic action is switched: degradation of the 2To whom correspondence should be addressed. E-mail: [email protected]. ′ 3 -terminated strand is down-regulated, whereas degradation of This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. the 5′-terminated strand is up-regulated (9, 13, 14). Upon 1073/pnas.1206081109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1206081109 PNAS | June 5, 2012 | vol. 109 | no. 23 | 8907–8912 Downloaded by guest on October 2, 2021 fi χ3 represent a group that displays relaxed recognition speci city right entry toward χ. The members of the first group define a recognition RecBCD χ3 RecBCD 3560 bp χ-containing helix structure for sequence-specific binding of ssDNA. The + χ ssDNA product 3 3140 bp members of the second group define an ionic latch structure dsDNA left entry 3 associated with the recognition helix. Our findings suggest that χ the latch is a χ-regulated structure responsible for controlling RecC mutation & Type “ ” 221 111 2 211 2 2 A a conformational switch that, we propose, opens a trap door to A A A A Enzyme 2 0 0 86H 05 χ 7 4 1 14 7 provide a new exit channel for the -containing ssDNA, thereby RecBC RecBCD L64A T Q38 W R R D133A R186A D136A D Q137A χ D705H avoiding nucleolytic degradation. Thus, recognition initiates min 05252 5 25252525 25 25 2525 2 5 2505 a conformational cascade in RecBCD that transforms its biological behavior by a unique mechanism that acts to divert ssDNA to an dsDNA alternative exit, thereby escaping nucleolytic demise. Results ssDNA χ-containing RecC-Channel Mutants Possess Approximately Wild-Type Levels of ssDNA product χ Helicase and Nuclease Activities. The core activities, dsDNA un- -containing 40±4 43±5 1±0.3 0.6±0.3 32±50.1±0.1 33±3 winding and nuclease, of the RecBCD mutants were examined; ssDNA product (%) 33±4 2±0.8 2±0.3 38±6 0.2±0.2 42±4 wild-type RecBCD and RecBC (which lacks nuclease activity) were used as the reference enzymes. All of the mutant enzymes Fig. 1. Type 1 (lost-recognition) mutants process dsDNA to produce negli- gible amounts of χ-specific ssDNA, whereas type 2 (relaxed-specificity) catalyzed unwinding of linear dsDNA, seen as disappearance of mutants display wild-type levels of χ recognition. Enzymes and reaction substrate and appearance of full-length ssDNA (Fig. S2). The times are indicated. The substrate was χ3 dsDNA [illustrated; processing mutants also possessed nuclease activity, which was manifest as produces ssDNA and χ-specific ssDNA fragments (only the χ-containing production of random-sized oligomeric ssDNA and, because of ssDNA product is shown)]. Final yields (± SD) of χ-containing ssDNA product the heterogeneous sizes, apparent loss of DNA. Because the are from at least three independent experiments.