DNA Damage and Tn7 Target Activation Page 1 1 2 DNA Damage

Genetics: Published Articles Ahead of Print, published on June 18, 2008 as 10.1534/genetics.108.088161 DNA damage and Tn7 target activation 1 2 3 DNA damage differentially activates regional chromosomal loci for Tn7 4 transposition in E. coli 5 6 7 Qiaojuan Shi, Adam R. Parks, Benjamin D. Potter, Ilan J. Safir, Yun Luo, Brian 8 M. Forster, Joseph E. Peters* 9 10 Department of Microbiology, Cornell University, Ithaca NY USA 11 12 13 14 15 16 17 18 19 20 21 22 23 *To whom correspondences should be addressed Page 1 DNA damage and Tn7 target activation 1 2 3 Running title: DNA damage and Tn7 target activation 4 5 Key Words: replication restart, target site selection, DNA double strand break 6 repair, chromosome structure 7 8 Corresponding authors contact information 9 175A Wing Hall 10 Ithaca, NY 14853 11 Ph.607-255-2271 12 FAX.607-255-3904 13 [email protected] 14 15 16 Page 2 DNA damage and Tn7 target activation 1 ABSTRACT 2 The bacterial transposon Tn7 recognizes replicating DNA as a target with 3 a preference for the region where DNA replication terminates in the Escherichia 4 coli chromosome. It was previously shown that DNA double strand breaks in the 5 chromosome stimulate Tn7 transposition where transposition events occur 6 broadly around the point of the DNA break. We show that individual DNA breaks 7 actually activate a series of small regional hotspots in the chromosome for Tn7 8 insertion. These hotspots are fixed and only become active when a DNA break 9 occurs in the same region of the chromosome. We find that the distribution of 10 insertions around the break is not explained by the exonuclease activity of 11 RecBCD moving the position of the DNA break, and stimulation of Tn7 12 transposition is not dependent on RecBCD. We show that other forms of DNA 13 damage, like exposure to UV light, mitomycin C or phleomycin also stimulate Tn7 14 transposition. However, inducing the SOS response does not stimulate 15 transposition. Tn7 transposition is not dependent on any known specific pathway 16 of replication fork reactivation as a means of recognizing DNA break repair. Our 17 results are consistent with the idea that Tn7 recognizes DNA replication involved 18 in DNA repair and reveals discrete regions of the chromosome that are 19 differentially activated as transposition targets. 20 Page 3 DNA damage and Tn7 target activation 1 INTRODUCTION 2 Mobile genetic elements have a profound effect on genome integrity in 3 bacteria. These elements have also been very important in the laboratory as 4 tools to understand the structure of the genome using genetic experiments that 5 complement microscopic analysis (Reviewed in (HIGGINS 2005)). We are using 6 the bacterial transposon Tn7 to better understand the nature of chromosome 7 replication, chromosome integrity, and chromosome evolution. 8 Tn7 uses one of its transposition pathways to target lagging-strand DNA 9 synthesis using the transposon-encoded target site selecting protein TnsE 10 (PETERS and CRAIG 2001a; PETERS and CRAIG 2001b). In addition to the TnsE 11 protein, transposition also requires a core transposition machinery consisting of 12 three proteins: TnsA, TnsB, and TnsC (TnsABC) (FLORES et al. 1990; KUBO and 13 CRAIG 1990; ROGERS et al. 1986; WADDELL and CRAIG 1988). Tn7 also controls 14 the orientation of transposition events in the chromosome where they occur in a 15 single left to right orientation with the direction of lagging-strand DNA replication 16 (PETERS and CRAIG 2001a). TnsABC+E transposition has a strong regional bias 17 for the area in the chromosome where DNA replication terminates. It was shown 18 that while TnsE actively directs transposition events to the region where DNA 19 replication terminates; transposition events with a mutant core machinery, TnsA, 20 TnsB, TnsCA225V (TnsABC*), that does not require a target site selecting protein 21 occur randomly across the E. coli chromosome. While active termination in the 22 terminus region focuses TnsABC+E transposition to a smaller region of the Page 4 DNA damage and Tn7 target activation 1 chromosome, active termination via the E. coli Tus replication termination protein 2 is not itself required for TnsE-mediated transposition (PETERS and CRAIG 2000). 3 In previous work it was shown that when DNA double strand breaks 4 (DSBs) were induced by the excision of Tn10, TnsABC+E transposition was 5 specifically stimulated (PETERS and CRAIG 2000). DSBs were induced through 6 the expression of a mutant form of the Tn10 transposase that causes the Tn10 7 element to excise, but not reinsert into a new target DNA (HANIFORD et al. 1989). 8 Tn10 excises through the formation of hairpins on the ends of the element 9 leaving a DSB in the donor DNA (KENNEDY et al. 1998), which can be repaired by 10 homologous recombination using a sister chromosome. Tn7 transposition events 11 that were stimulated by the DSB were distributed broadly around the break for 12 many thousands of base pairs, not at the point of the break. The observation 13 that DSBs activated the region proximal to the break as a transposition target 14 suggested that TnsE recognized a facet of DSB repair (SMITH 2001). 15 Paradoxically, the distribution of transposition events differed greatly with the two 16 positions where DSBs were induced in this study (PETERS and CRAIG 2000). 17 We investigated what factors are responsible for stimulating TnsE- 18 mediated transposition as a response to DNA damage. We find that DSBs 19 activate transposition into discrete spans in the chromosome that we call 20 “hotspots.” These hotspots are at fixed positions and are only activated by DSBs 21 in the same general region of the chromosome. The stimulatory effect of DSBs 22 does not require the RecBCD enzyme that normally processes DSBs. We find 23 that multiple types of DNA damage stimulate TnsABC+E transposition, but this is Page 5 DNA damage and Tn7 target activation 1 not explained by a specific interaction with one of the replication fork restart 2 proteins or SOS induction. Our data are consistent with a model where TnsE is 3 capable of recognizing DNA replication involved in the repair of DNA DSBs. 4 5 MATERIAL AND METHODS 6 Bacterial strains and plasmids: Strains were constructed using standard 7 P1 transduction methods (MILLER 1992; PETERS 2007) (Table 1). All Tn10 8 insertion alleles were confirmed in the final strain by DNA sequencing a template 9 made using arbitrary PCR (see below). Drug makers were removed from frt- 10 containing constructs with the FLP recombinase produced from pCP20, which 11 was subsequently lost at 42° (DATSENKO and WANNER 2000). Transduction of the 12 dnaC809 allele was by linkage to thrA-34::Tn10 and confirmed by PCR 13 amplification followed by restriction digestion with HinFI (the dnaC809 mutation 14 abolishes a HinFI site within the dnaC gene)(SANDLER et al. 1996). Strain AP457 15 was constructed by the method described by Datsenko and Wanner (DATSENKO 16 and WANNER 2000). Briefly, primers JEP191 (5’-AGA AAA ACT CAT CGA GCA 17 TCA AAT GAA ATG AAA CTG CAA TTT ATT CAT A GT GTA GGC TGG AGC 18 TGC TTC-3’) and JEP192 (5’-ATG AGC CAT ATT CAA CGG GAA ACG TCT 19 TGC TCG AGG CCG CGA TTA AAT TCA TAT GAA TAT CCT CCT TAG-3’) 20 were used to amplify the CamR gene (and flanking frt sites) from plasmid pKD3 21 with 40 bp homology to KanR gene cassettes. The PCR product was crossed 22 into AP429 carrying pKD46, which was induced with 0.2% arabinose and the 23 plasmid subsequently lost by growth at 42˚ giving AP453. Strain AP699 Page 6 DNA damage and Tn7 target activation 1 (BW25113 attTn7::frt-CamR-frt) was made in a similar fashion with primers 2 JEP295 (5’-ATG TTT TTA ATC AAA CAT CCT GCC AAC TCC ATG TGA CAA 3 AGT GTA GGC TGG AGC TGC TTC-3’) and JEP196 (5’-TCA GTC TGA TTT 4 AAA TAA GCG TTG ATA TTC AGT CAA TTA CCA TAT GAA TAT CCT CCT 5 TAG- 3’). This amplicon was used to cross the CamR marker into a site 59 bp 6 downstream of attTn7 without loss of host sequence. The ∆sbcDC::dhfr allele 7 was also constructed by the method described by Datsenko and Wanner 8 (DATSENKO and WANNER 2000). Primers JEP126 (5’ – CCA TGC TTT TTC GCC 9 AGG GAA CCG TTA TGC GCA TCC TTC ACA CCT CAG ACC TGG AAA GTA 10 CGT TTG CAG TGA AAT AAC TAT TCA GCA GGA TAA TGA ATA CGC TAT 11 TTA GGT GAC ACT ATA G – 3’) and JEP127 (5’ – GTA TTC ATT ATC CTG 12 CTG AAT AGT TAT TTC ACT GCA AAC GTA CTT TCC AGT AAT ACG ACT 13 CAC TAT AGG G- 3’) were used to amplify a dhfr gene that imparts resistance to 14 trimethoprim. The resulting PCR fragment was crossed into the chromosome as 15 described above. PCR analysis with flanking primers and DNA sequencing 16 confirmed that all, but the most 5’ five amino acids of coding information of sbcD 17 through the most 3’ five amino acids of coding information of sbcC were replaced 18 with the dhfr cassette. Plasmid vectors were constructed with standard 19 techniques as described (SAMBROOK et al. 1989)(Table 2). 20 Transposition assay: A promoter capture assay, called the papillation 21 assay, monitors transposition levels in lawns of bacteria on indicator media 22 (HUISMAN and KLECKNER 1987; STELLWAGEN and CRAIG 1997a). A miniTn7 23 element encoding the lactose utilization genes without the requisite promoter is Page 7 DNA damage and Tn7 target activation 1 situated in the tester strain attTn7 site (JP617 or AP457) (Table 1).

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