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The Bacillus Subtilis Addab Helicase/Nuclease Is Regulated by Its Cognate Chi Sequence in Vitro

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The Bacillus Subtilis Addab Helicase/Nuclease Is Regulated by Its Cognate Chi Sequence in Vitro doi:10.1006/jmbi.2000.3556 available online at http://www.idealibrary.com on J. Mol. Biol. (2000) 298, 7±20 The Bacillus subtilis AddAB Helicase/Nuclease is Regulated by its Cognate Chi Sequence in Vitro FreÂdeÂric CheÂdin1,2, S. Dusko Ehrlich2 and Stephen C. Kowalczykowski1* 1Sections of Microbiology and The AddAB enzyme is important to homologous DNA recombination in of Molecular and Cellular Bacillus subtilis, where it is thought to be the functional counterpart of the Biology, University of RecBCD enzyme of Escherichia coli. In vivo, AddAB responds to a speci®c California, Davis, CA 95616- ®ve-nucleotide sequence (50-AGCGG-30 or its complement) in a manner 8665, USA analogous to the response of the RecBCD enzyme to interaction with w sequences. Here, we show that puri®ed AddAB enzyme is able to load at 2Laboratoire de GeÂneÂtique a double-stranded DNA end and is both a DNA helicase and nuclease, Microbienne INRA, Domaine whose combined action results in the degradation of both strands of the de Vilvert, 78352, Jouy en DNA duplex. During translocation, recognition of the properly oriented Josas, Cedex, France sequence 50-AGCGG-30 causes attenuation of the AddAB enzyme nucle- ase activity that is responsible for degradation of the strand 30-terminal at the entry site. Therefore, we conclude that 50-AGCGG-30 is the B. subtilis Chi site and it is hereafter referred to as wBs. After encountering wBs, both the degradation of the 50-terminal strand and the helicase activity persist. Thus, processing of a double-stranded DNA end by the AddAB enzyme produces a duplex DNA molecule with a protruding 30-terminated single-stranded tail, a universal intermediate of the recombination process. # 2000 Academic Press *Corresponding author Keywords: Chi; nuclease; homologous recombination; helicase; AddAB Introduction cal for the initiation of this repair pathway (for a review, see Smith, 1988; Kowalczykowski et al., The repair of genotoxic lesions is a central aspect 1994). RecBCD enzyme will bind to dsDNA ends of DNA metabolism in living cells, and many com- and proceed to unwind the DNA while extensively 0 plex enzymatic processes are devoted to maintain- degrading the strand terminating 3 at the entry ing the integrity of the genome. Homologous site (referred to as the ``top-strand''; see Figure 1) recombination is one important pathway by which (Dixon & Kowalczykowski, 1993). This destructive potentially lethal lesions, such as double-stranded mode, however, is switched to a recombinogenic DNA breaks, can be repaired. These lesions arise mode after the enzyme recognizes a pro- frequently during normal cell growth if DNA repli- perly oriented w sequence. The w octamer, cation is impeded (Michel et al., 1997) or, in eukar- 50-GCTGGTGG-3', initially de®ned as a recBC- yotes, at the onset of meiosis (Baudat & Nicolas, dependent recombination hotspot (Lam et al., 1974; 1997; Dernburg et al., 1998; McKim & Hayashi- Smith et al., 1981; Stahl et al., 1975), elicits a num- Hagihara, 1998; Sun et al., 1989). In Escherichia coli, ber of biochemical changes in RecBCD enzyme's double-stranded DNA (dsDNA) breaks are behavior: the enzyme brie¯y pauses at w before repaired by homologous recombination between unwinding resumes, at which point the top-strand the damaged DNA and an intact copy of the nuclease is down-regulated, and a weaker nuclease chromosome. Numerous genetic and biochemical activity is activated on the opposite strand data have shown that the RecBCD enzyme is criti- (Anderson & Kowalczykowski, 1997a; Dixon & Kowalczykowski, 1991, 1993). These modi®cations produce a processed dsDNA with a 30 single- Abbreviations used: ssDNA, single-stranded DNA; dsDNA, double-stranded DNA; SSB protein, single- stranded DNA (ssDNA) tail terminating at w that stranded DNA-binding protein. can be bound by the RecA protein to initiate the E-mail address of the corresponding author: homologous pairing phase of genetic recombina- [email protected] tion. The formation of a RecA ®lament on ssDNA 0022-2836/00/010007±14 $35.00/0 # 2000 Academic Press 8 AddAB-Chi Interactions in Vitro Figure 1. Four potential wBs- speci®c fragments can be produced upon interaction of the AddAB enzyme with wBs-containing DNA. The region of the DNA between wBs and the enzyme's entry point is referred to as the upstream region while the region past wBs to the opposite end of the DNA is the downstream region. The top-strand is the 30-terminating strand at the entry point; the opposite strand is referred to as the bottom-strand. The various putative wBs-fragments are indicated. is a crucial step in this initiation reaction, and the to interact with an enzyme possessing both heli- w-activated RecBCD enzyme is directly responsible case and exonuclease activities. In H. in¯uenzae, for coordinating the loading of the RecA protein this enzyme is a clear homolog of RecBCD. How- onto w-containing ssDNA. This ensures maximal ever, for the two Gram-positive bacteria L. lactis participation of this w-containing ssDNA in the and B. subtilis, the corresponding enzymes seem to process of genetic recombination (Anderson & belong to a novel class of enzymes that are func- Kowalczykowski, 1997b). tionally, but not structurally, homologous to the The interaction between the E. coli RecBCD RecBCD class of enzymes. enzyme and w has been extensively studied, and it In B. subtilis, the model organism for Gram-posi- serves as a model for understanding how homolo- tive bacteria, the counterpart of the RecBCD gous DNA recombination is initiated in bacteria. enzyme is thought to be the AddAB enzyme. However, until very recently, evidence for conser- Genetically, mutations in either addA or addB genes vation of an interaction between a helicase/exonu- result in reduced cell viability, increased sensitivity clease and a w-like regulatory sequence as a means to DNA-damaging agents, and reduced levels of to initiate homologous recombination was strik- DNA recombination in certain strains (Kooistra ingly sparse. RecBCD homologs have been found et al., 1988; Alonso et al., 1988), which are pheno- in several enterobacteria as well as in other, more typic characteristics of recB or recC mutations in distantly related bacteria, such as Haemophilus E. coli. In addition, expression of AddAB enzyme in¯uenzae, Pseudomonas aeruginosa, Neisseria menin- in E. coli complements RecBC phenotypes gitidis and Mycobacterium tuberculosis. However, (Kooistra et al., 1993) and, in vitro, the AddAB RecBCD-w interactions, as de®ned by the pro- enzyme possesses exonuclease and helicase activi- duction of a diagnostic w-speci®c fragment by the ties (Haijema et al., 1996a). From previous in vivo E. coli enzyme, seem to be conserved only in work, we showed that a speci®c ®ve-nucleotide enteric bacteria that are closely related to E. coli sequence, 50-AGCGG-30 allowed formation of a (McKittrick & Smith, 1989). This suggests that high molecular weight form of plasmid DNA, pre- either the RecBCD-w interaction is restricted to a sumably by protecting this DNA from degradation small group of microbes or that sequences other by AddAB (CheÂdin et al., 1998). Although hotspot than the canonical w sequence might be used in activity was not demonstrated for this sequence, it different organisms. This latter view is supported was proposed that this pentamer was the B. subtilis by the recent identi®cation of speci®c short DNA analog of the well-described Chi site of E. coli (wEc). sequences (5-8 bp) that protect dsDNA from exo- Below, we establish that this supposition was cor- nucleolytic degradation in the bacteria Lactococcus rect, and 50-AGCGG-30 will therefore be referred to lactis (Biswas et al., 1995), H. in¯uenzae (Sourice as wBs. et al., 1998) and Bacillus subtilis (CheÂdin et al., 1998). Despite these parallels with the E. coli system, In all cases, those putative w sequences are believed the biochemical consequences of the interaction AddAB-Chi Interactions in Vitro 9 between AddAB enzyme and its putative wBs concentrations (Mg(OAc)2). As expected, the initial sequence are unclear. Although the in vivo obser- dsDNA is rapidly processed by the enzyme and is vations suggest that the enzyme loses its ability to converted to full-length ssDNA and a variety of degrade double-stranded DNA after wBs is recog- degradation products. The amount of full-length nized (CheÂdin et al., 1998), the exact nature of the ssDNA, however, varied strongly with the concen- changes elicited by this interaction could not be tration of magnesium ions. At 0.5 mM Mg(OAc)2, inferred. Here, we show that the puri®ed AddAB about 80 % of the unwound DNA was full-length enzyme is a potent helicase and exonuclease ssDNA after ®ve minutes, whereas at 2 mM which, in contrast to the RecBCD enzyme, Mg(OAc)2, only 10 % of the unwound DNA was degrades both strands of the DNA duplex almost full-length. Similar results were obtained with symmetrically. When a properly oriented wBs dsDNA that was linearized with other restriction sequence is present, exonucleolytic degradation of endonucleases or with dsDNA that was 30-end the 30-terminated strand at the entry point ceases, labeled (data not shown). These results show that, while degradation of the 50-terminated strand con- at low magnesium ion concentrations, the nuclease tinues. This alteration in the behavior of the activity of AddAB enzyme is low, so that proces- enzyme results in the production of a dsDNA mol- sing of linear dsDNA under these conditions ecule carrying a long 30-overhang, an intermediate mostly produces fully unwound ssDNA. However, that is universal for the initiation of DNA recombi- at higher magnesium ion concentrations, AddAB nation. Our data also imply that the interaction both unwinds and degrades dsDNA, as evidenced between an exonuclease/helicase and a regulatory by the decrease in production of full-length sequence is an evolutionarily conserved means to ssDNA.
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