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The Recombination Hot Spot X Is a Regulatory Element That Switches the Polarity of DNA Degradation by the Recbcd Enzyme

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The Recombination Hot Spot X Is a Regulatory Element That Switches the Polarity of DNA Degradation by the Recbcd Enzyme Downloaded from genesdev.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press The recombination hot spot X is a regulatory element that switches the polarity of DNA degradation by the RecBCD enzyme Daniel G. Anderson I and Stephen C. Kowalczykowski 1-3 1Genetics Graduate Group, 2Sections of Microbiology and of Molecular and Cellular Biology, University of California at Davis, Davis, California 95616-8665 USA Homologous recombination in Escherichia coli is stimulated at DNA sequences known as X sites. Stimulation requires the multifunctional RecBCD enzyme, which is both a helicase and a 3' ~ 5' exonuclease. Upon recognition of a properly oriented X site, the 3' -9 5' exonuclease activity is attenuated. Here we show that in addition to attenuation of the 3' ~ 5' exonuclease activity, recognition of X by the RecBCD enzyme also up-regulates a nuclease activity of the opposite polarity, resulting in an enzyme that now preferentially degrades 5' ~ 3'. These results demonstrate that X is a unique regulatory element that converts the antirecombinogenic form of the RecBCD enzyme into a recombinogenic form by causing two distinct enzymatic changes: attenuation of the 3' ~ 5' nuclease activity, and up-regulation of the 5' ~ 3' nuclease activity. The consequence of X recognition is the production of a recombination intermediate possessing a 3'-ssDNA overhang terminating at the X sequence. This processing of a dsDNA end to a 3'-ssDNA overhang parallels that which occurs during the initation of homologous recombination in other pathways in E. coli, and in other organisms such as the yeast Saccharomyces cerevisiae. [Key Words: Recombination; RecBCD; X; nuclease; helicase; regulation] Received December 4, 1996; revised version accepted January 30, 1997. The RecBCD enzyme is essential for the main pathway ceeds to unwind the DNA duplex while simultaneously of homologous recombination in Escherichia coli. This degrading the DNA from this end (Telander-Muskavitch heterotrimeric enzyme is composed of products of the and Linn 1981; Taylor and Smith 1985). Degradation of reck recC, and recD genes (for review, see Smith 1988; the DNA is asymmetric, with the 3'-terminal strand Kowalczykowski et al. 1994). Inactivation of either the relative to the entry site of the RecBCD enzyme being recB or recC genes leads to a reduction of homologous degraded much more extensively than the 5'-terminal recombination by up to 1000-fold in vivo (Howard- strand (Dixon and Kowalczykowski 1993). Flanders and Theriot 1966; Emmerson 1968). Homologous recombination in wild-type Escherichia The RecBCD enzyme possesses a number of different coli is stimulated at cis-acting DNA sequences known as nucleolytic activities; it is an ATP-dependent exonucle- X sites (5'-GCTGGTGG-3')(Lam et al. 1974; Stahl et al. ase and an ATP-stimulated single-stranded DNA 1975; Smith et al. 1981). In vivo, these DNA elements (ssDNA) endonuclease (Telander-Muskavitch and Linn stimulate homologous recombination 5- to 10-fold in a 1981; Taylor 1988; Smith 1990; Kowalczykowski et al. recBCD-dependent manner. Stimulation by x is polar, 1994). In addition to these degradative properties, the with maximum stimulation at x and decaying down- RecBCD enzyme is a highly processive helicase, capable stream relative to the entry site of the RecBCD enzyme of unwinding up to 30,000 bp per binding event at rates (Stahl et al. 1980; Ennis et al. 1987; Cheng and Smith of up to 1000 bp/sec (Roman and Kowalczykowski 1989; Myers et al. 1995a, b). The polar nature of X stimu- 1989a; Roman et al. 1992; Eggleston and Kowalc- lation in vivo is a direct result of the dramatic changes in zykowski 1993). The preferred DNA substrate for the the RecBCD enzyme function that take place after x rec- RecBCD enzyme is a double-stranded DNA (dsDNA) ognition. When × is recognized by a translocating end, to which it binds with high affinity and then pro- RecBCD enzyme molecule, DNA degradation halts -4-5 nucleotides upstream of the x site, after which the en- zyme continues to unwind the DNA with its 3' ~ 5' SCorresponding author. exonuclease activity greatly attenuated (Ponticelli et al. E-MAIL [email protected]; FAX (916) 752-5939. 1985; Dixon and Kowalczykowski 1993; Taylor and GENES & DEVELOPMENT 11:571-581 © 1997 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/97 $5.00 571 Downloaded from genesdev.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press Anderson and Kowalczykowski Smith 1995). The continued unwinding of the dsDNA opposite end of the DNA to be the downstream region downstream of X leads to production of a ssDNA tem- (see Fig. 1). In addition, the strand of dsDNA that termi- plate that can now be utilized by the homologous pairing nates 3' at the entry point of the RecBCD enzyme is and strand-exchange protein RecA (Dixon and Kowalc- defined as the top strand. The DNA strand opposite the zykowski 1991). top strand is defined as the bottom strand. Thus, the Perhaps the single most essential enzyme for homolo- unwinding and specific cleavage of DNA containing a X gous recombination in E. coli is the RecA protein. Mu- site by the RecBCD enzyme has the potential to generate tation of the recA gene causes a 10 4- to 10S-fold reduc- four distinct X-specific ssDNA fragments: top strand, up- tion in homologous recombination (Clark and Margulies stream fragment; top strand, downstream fragment; bot- 1965). In vitro, this protein is able to promote pairing and tom strand, upstream fragment; and bottom strand, exchange between homologous DNA substrates (for re- downstream fragment. To establish whether the DNA view, see Kowalczykowski and Eggleston 1994). One re- fragment observed by Dixon and Kowalczykowski (1995) quirement for RecA protein-promoted pairing in vitro is was X-specific and derived from the bottom strand, un- that at least one of the DNA substrates must possess winding reactions using linear pBR322 x+F (Fig. 2A, B) some ssDNA character. This is in contrast to the obser- were compared to those using linear pBR322 x+E (Fig. vation that the majority of recombination events which 2C), both of which contain a single × site at a different occur in vivo, and in particular those that are mediated location in pBR322. If the production of this new 5'-end- by the RecBCD pathway, involve donor DNA substrates labeled species is X-specific, then the size of this species that are double-stranded (for review, see Kowalc- should change as the location of × is varied. In addition, zykowski et al. 1994). The RecBCD enzyme acts to ini- these reactions were compared to unwinding reactions tiate homologous recombination by processing dsDNA using linear pBR322, which contains no × sites (Fig. 2D). into a ssDNA substrate suitable for RecA protein action Figure 2A shows the products of an unwinding reac- (Taylor and Smith 1985; Roman and Kowalczykowski tion using linearized pBR322 x+F, which was 5'-end-la- 1989b; Dixon and Kowalczykowski 1991). beled. The efficiency of X recognition by the RecBCD Although the ability of × to attenuate the 3' --~ 5' exo- enzyme is only -20% (Taylor et al. 1985; Dixon and nuclease of the RecBCD enzyme has been demonstrated, the effect of × recognition on the weaker 5' ~ 3' exo- nuclease activity is unclear (Dixon and Kowalczykowski 1993). In this paper we directly demonstrate that recog- Upstream nition of × results in the up-regulation of the 5'~ 3' Top Strand ,~ Chi~ ~ RecBCD exonuclease activity, in addition to the attenuation of 5' ~ 3' the 3' ~ 5' exonuclease activity. Thus, the consequence 3' 5' 4 of × recognition is the production and net preservation of Bottom Strand ~ t a recombination intermediate possessing a 3'-ssDNA Downstream overhang terminating at the X sequence. This processing of a dsDNA end to a 3'-ssDNA overhang parallels that which occurs in other pathways of homologous recom- bination in E. coli and in other organisms such as the yeast S. cerevisiae (for review, see Kowalczykowski et al. Top-strand, downstream Top-strand, upstream 1994). X-specific fragment X-specific fragment K 7 X 5' I ~ 3' Results + 3' 5' Recognition of X leads to the production of X-specific DNA fragments derived from both strands of dsDNA Bottom-strand, downstream Bottom-strand,upstream As mentioned previously, recognition of × by a trans- ~-specific fragment ~-specific fragment locating RecBCD enzyme molecule leads to attenuation Figure 1. Possible x-specific fragments produced by the inter- of 3' -~ 5' exonuclease activity. The continued unwind- action of the RecBCD enzyme with x-containing DNA. The ing of the DNA after × produces a downstream ssDNA region of dsDNA between X and the entry site for the RecBCD fragment of a length corresponding to the distance be- enzyme is referred to as the upstream region, and the region past tween × and the distal end of the DNA (Dixon and Ko- Xto the opposite end of the DNA is the downstream region. The walczykowski 1993). In addition, Dixon and Kowalczy- strand of DNA that terminates 3' at the entry site of the kowski (1995) observed that another 5'-end-labeled DNA RecBCD enzyme is termed the top-strand; the opposite strand is fragment was created, possibly corresponding to the re- referred to as the bottom-strand. The combined nuclease and helicase action of the RecBCD enzyme on a X-containing gion of DNA that is upstream of × but derived from the dsDNA substrate has the potential to generate four x-specific opposite DNA strand. For clarity, we define the region ssDNA fragments: top-strand, upstream X-specific fragment; between the × site and the end of the DNA at which the bottom-strand, upstream X-specific fragment; top-strand, down- RecBCD enzyme initiated unwinding to be the upstream stream X-specific fragment; and bottom-strand, downstream region, and the DNA between the X site and the X-specific fragment.
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