Genetics: Published Articles Ahead of Print, published on June 18, 2008 as 10.1534/genetics.108.088161
DNA damage and Tn7 target activation
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3 DNA damage differentially activates regional chromosomal loci for Tn7
4 transposition in E. coli
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7 Qiaojuan Shi, Adam R. Parks, Benjamin D. Potter, Ilan J. Safir, Yun Luo, Brian
8 M. Forster, Joseph E. Peters*
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10 Department of Microbiology, Cornell University, Ithaca NY USA
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23 *To whom correspondences should be addressed
Page 1 DNA damage and Tn7 target activation
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3 Running title: DNA damage and Tn7 target activation
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5 Key Words: replication restart, target site selection, DNA double strand break
6 repair, chromosome structure
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8 Corresponding authors contact information
9 175A Wing Hall
10 Ithaca, NY 14853
11 Ph.607-255-2271
12 FAX.607-255-3904
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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.
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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.
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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). In this
2 configuration the lac genes are not transcribed and the strain is unable to use
3 lactose (Lac-). If the element transposes to a new position in the correct
4 orientation in an active gene the element will be transcribed resulting in a
5 phenotypically Lac+ bacterium. On MacConkey’s lactose indicator media or on
6 LB lactose X-gal media, Lac+ cells form differentially colored microcolonies, or
7 papillae, on the lawn of Lac- cells. The number of Lac+ papillae provides a
8 measure of transposition levels. To rule out any effect of the tested allele or
9 condition on cell viability or papillae formation an internal control is always tested
10 where papillae formation is compared with a background strain that lacks TnsE.
11 Transposon mapping: To map Tn7 transposon insertion events, the Lac+
12 papillae were purified and P1 linkage mapping was used to determine if
13 transposition had occurred or if an unrelated Lac+ mutation occurred, as
14 described previously using strain JP949 or AP724 (PETERS and CRAIG 2000).
15 For each Tn10 DSB position, two Lac+ events were chosen at random from each
16 of the 12 experiments. Two Lac+ events were chosen at random from each of 24
17 experiments from cells expressing TnsABC+E following UV irradiation. Because
18 the Lac+ events that result from recombination and not Tn7 transposition varies
19 from 30 to 60%, the total number of real Tn7 transposition events that were
20 mapped is not equal. Tn7 transposition events were mapped by sequencing from
21 the left and/or right ends of products made using arbitrary PCR as described
22 previously and the position in the genome determined using the E. coli MG1655
23 genome sequence version M52 (BLATTNER et al. 1997; PETERS and CRAIG
Page 8 DNA damage and Tn7 target activation
1 2001a). In ~5% of the cases both the left and right ends were sequenced to
2 ensure that real transposition occurred. Tn10 insertions were mapped using
3 arbitrary PCR as above, but replacing the arbitrary primer (JEP293 5’–GGC CAC
4 GCG TCG ACT AGT CAN NNN NNN NNN GAT CG-3’) and the element specific
5 primers #1 and #2 from (NICHOLS et al. 1998). Primer #2 was used for
6 sequencing the final Tn10 template.
7 β-galactosidase assay: The β-galactosidase activity was measured as
8 described in triplicate for each sample (MILLER 1992).
9 DNA damage induction: To determine the effect of DSBs, a mutant Tn10
10 transposase (pZT383-TnpP167S) was expressed that allows the element to excise,
11 but not to insert (GALITSKI and ROTH 1997; HANIFORD et al. 1989; PETERS and
12 CRAIG 2000). To ensure that the effect was from the DNA break and not an
13 unrelated effect of the transposase, control experiments utilized the same vector
14 with mutations in the active site of the Tn10 transposase (pJP135-
D97A/D161A 15 Tnp )(Table 2)(PETERS and CRAIG 2000). Plates were exposed to 10
16 millijoules of UV in a Stratalinker 1800 (Stratagene). Mitomyocin C (Sigma) and
17 phleomycin (Sigma) were diluted in water and added to the media prior to
18 pouring after it cooled.
19
20 RESULTS
21 Our objective is to understand processes that contribute to the distribution
22 of Tn7 transposition events around induced DSBs at specific locations in the
23 chromosome. It was previously shown that a very different distribution of Tn7
Page 9 DNA damage and Tn7 target activation
1 insertions was found when DSBs were induced at two different positions in the E.
2 coli chromosome (PETERS and CRAIG 2000). One simple model for why
3 transposition events might occur at some distance from the original position of an
4 induced DSB is that the potent exonuclease activity of the RecBCD enzyme
5 could move the actual genetic position of the DSB. The extent to which the
6 RecBCD enzyme would process DNA is a function of the density of properly
7 oriented chi sties around the point of a DSB. Chi sites are polar, eight base pair
8 sequences that alter the exonuclease activity and RecA-loading activity of the
9 RecBCD complex (KOWALCZYKOWSKI 2000; KUZMINOV 1999). Given that the
10 distribution of chi sites varies in the chromosome, it seemed possible that chi site
11 density could explain differences in the distribution of transposition events around
12 DSBs (See for example, (SMITH 2001)). This was of special interest because,
13 fortuitously, one of the original DSB positions (srlD-3131::Tn10, 2826 kb) was in
14 a region that was very sparse for chi sites (J. E. Peters, unpublished). To
15 address this idea we analyzed the distribution of TnsABC+E transposition events
16 when DSBs were induced at three positions that were not examined previously,
17 zbi-29::Tn10, recD-1901::Tn10, and yhfT-3084::Tn10 (MATERIALS AND
18 METHODS). Tn7 transposition was monitored through the use of a promoter
19 capture assay using a Tn7 derivative that contains the lactose utilization genes
20 without a promoter. Transposition of the Tn7 derivative can allow the host to
21 acquire the ability to grow on lactose (Lac+) if the element moves to a transcribed
22 region of the chromosome. On indicator media a Lac+ cell will form a
23 differentially colored microcolony, or papilla, on the lawn of bacteria. The number
Page 10 DNA damage and Tn7 target activation
1 of papillae provides a measure of the frequency of transposition. DSB induction
2 at any of the sites chosen specifically stimulated TnsABC+E transposition (Figure
3 1A and Table 3). As found previously, a background level of Lac+ papillae
4 always occurs in the assay that results from genetic events unrelated to Tn7
5 transposition (Figure 1A and Table 3)(PETERS and CRAIG 2000).
6 We examined the DNA sequence between each Tn10 allele used to
7 induce DSBs and the resulting Tn7 insertion events that were stimulated by the
8 DSB for all the data shown in this paper. In each case we paid particular
9 attention to whether the Tn7 insertions occurred on the end of the break closest
10 to oriC or closest to terC to judge the contribution of chi sites in the chromosome
11 (Table 4). We also analyzed the data from two Tn10 alleles used in previously
12 published work (see figure 6 in (PETERS and CRAIG 2000)). While noting the
13 number of chi sites and the distance between the position of the DSB and the
14 first Tn7 transposition events in both directions; we arbitrarily ignored events that
15 were over 300kb from the DSB and stopped considering chi sites after 20
16 correctly oriented sites (chi sites are recognized only 20% of the time in vivo and
17 in vitro (DIXON et al. 1994; DIXON and KOWALCZYKOWSKI 1993; KUZMINOV et al.
18 1994), therefore, 20 chi sites would stop 99% of the RecBCD enzymes )(Table
19 4). In examining the distribution of TnsE-mediated transposition events that are
20 stimulated by the DSB, we find no obvious relationship between the frequency of
21 chi sites and the relative distribution of insertions around the DSB (Table 4).
22 While no chi sites were found between the induced DSB and the first insertions in
23 some cases (i.e. srlD-3131::Tn10), in other cases, many chi sites were found
Page 11 DNA damage and Tn7 target activation
1 before the first insertions proximal to the DSB (Table 4). Interestingly, TnsE-
2 mediated insertion events still occurred broadly around the DSB in the recD-
3 1901::Tn10 strain. The RecBCD enzyme complex is responsible for tailoring
4 DNA double-strand breaks and loading RecA for their repair. In a recD strain
5 background the RecBC+(D-) enzyme will not degrade from the initial DSB position
6 (AMUNDSEN et al. 1986; CHAUDHURY and SMITH 1984), but will immediately load
7 RecA for replication-mediated DSB repair (KUZMINOV and STAHL 1999)(see
8 discussion).
9 We tested if a specific interaction between TnsE and the RecBCD
10 complex was required for DSBs to stimulate transposition. We took advantage of
11 the previous finding that the RecFOR proteins can load RecA at DSBs in certain
12 suppressor backgrounds. A RecFOR-dependent DSB repair pathway that is
13 independent of RecBCD can be activated in sbcB sbcC sbcD strains (CLARK and
14 SANDLER 1994). We determined if inducing DSBs with the srlD3131::Tn10 would
15 stimulate TnsE-mediated transposition in a ∆recC background that also
16 contained the ∆sbcB ∆sbcC ∆sbcD alleles (PETERS and CRAIG 2000)
17 (MATERIALS AND METHODS). In these experiments we monitored Tn7
18 transposition on LB lactose X-Gal plates given the observation that recC strains
19 are sensitive to bile salts found in MacConkey’s media (PRIETO et al. 2006). As
20 expected, we found that the ∆recC allele causes the strain to be sensitive to UV
21 light and mitomycin C, but UV and mitomycin C resistance is reestablished in the
22 ∆recC ∆sbcB ∆sbcC ∆sbcD strain (data not shown). We found that TnsE-
23 mediated transposition increased significantly (p<0.05 from a t-test, two tailed,
Page 12 DNA damage and Tn7 target activation
1 unequal variance) when DSBs were induced in the wild type or ∆recC ∆sbcB
2 ∆sbcC ∆sbcD strains, but no significant change was found in the ∆recC
3 background (Figure 2). Tn7 transposition in the ∆recC ∆sbcB ∆sbcC ∆sbcD strain
4 was not stimulated to the same extent as was found in the rec+ sbc+ background.
5 This result may be explained by a lower efficiency of DSB repair in the ∆recC
6 ∆sbcB ∆sbcC ∆sbcD strain as compared to the wild type strain. This effect is
7 likely compounded in the strains with the TnsABC+E proteins because two DSBs
8 are actually formed, one by the Tn10 and a second DSB at the original
9 chromosomal position of the Tn7 element. These results indicate that the ability
10 of DSBs to stimulate TnsE-mediated transposition is not dependent on an
11 interaction with the RecBCD proteins nor a specific DNA structure made with this
12 enzyme complex.
13 Induced DSBs activate transposition hotspots in the chromosome:
14 The analysis of multiple DSB positions revealed an interesting finding. TnsE-
15 mediated transposition events seemed to occur at hotspots in the chromosome
16 that appeared to be activated by individual DSBs (Figures 1B-D). We use the
17 term hotspot to describe when multiple insertions occur within a region of less
18 than about a kb of one another in the 4.6 Mbp E. coli chromosome. However, it
19 is important to note that we never found an example where the same insertion
20 site was used (i.e. these are small regional hotspots for insertion). As shown
21 below, these regional hotspots are not explained by a selection bias for highly
22 expressed genes. One of the clearest examples of a hotspot that appeared to be
23 stimulated as a response to a DSB occurred when a break was induced at 3504
Page 13 DNA damage and Tn7 target activation
1 kb on the chromosome with yhfT-3084::Tn10 (Figures 1B and 3A). 36% (4/11) of
2 the TnsE-mediated transposition events that were stimulated by the break
3 occurred within a small ~500 bp patch of sequence that was almost 160 kb away
4 from the yhfT-3084::Tn10 (Figures 1B and 3A). In another example, 27% (4/15)
5 of the transposition events that were stimulated by inducing breaks at 2949 kb on
6 the chromosome with recD-1901::Tn10 occurred in a ~800 base pair region that
7 was over 27 kb away from the DSB (Figure 1C). It is of additional interest to
8 consider the incidence and distribution of transposition hotspots in the recD-
9 1901::Tn10 strain (see discussion). All positions where DSBs were induced
10 seemed to activate one or more transposition hotspots (Figure 1). This was also
11 found to be true when we re-examined the TnsE-mediated transposition events
12 collected in an earlier work (PETERS and CRAIG 2000)(Table 4). By comparing
13 the Tn7 insertions that resulted from inducing DSBs at 698 kb (zbf-
14 3057::Tn10)(PETERS and CRAIG 2000) and 820 kb (zbi-29::Tn10)(Figure 1D) we
15 found something especially intriguing; DSBs at both of these positions caused a
16 transposition hotspot around the gene serW (925 kb), even though this gene was
17 227 kb and 106 kb from the position of the DSBs, respectively (Table 4). This
18 observation suggested that the hotspots were at fixed positions in the
19 chromosome and could be activated by DSBs at many positions.
20 Hotspots are fixed in the chromosome and are only activated by
21 DSBs in the same general region of the chromosome: To confirm the idea
22 that the hotspots were at fixed positions, we examined the effect of inducing
23 DSBs at different distances (157, 83, and 19 kb) from the most active
Page 14 DNA damage and Tn7 target activation
1 transposition hotspot, yrbL-mtgA (3346-3347)(Figure 3A and 4). We
2 reconstructed the strain where DSBs were induced using the yhfT-3084::Tn10 at
3 3504 kb in the chromosome and retested this strain to rule out any unknown
4 factors related to the original construction (Figure 4A). We found that 35%
5 (6/17) of the insertions again occurred in the yrbL-mtgA hotspot which was 157
6 kb away (Figures 3A and 4A). When we induced DSBs in smg (smg-
7 3082::Tn10), a position that was 83 kb from the yrbL-mtgA hotspot, we found that
8 55% (6/11) of the insertions occurred in the yrbL-mtgA hotspot (Figures 3A and
9 4B). When we induced DSBs in b3219 (b3219-6::Tn10), a position 19 kb from the
10 yrbL-mtgA hotspot, there seemed to be a drop in the usage of the yrbL-mtgA
11 hotspot; one in 13 insertions occurred in the hotspot (Figures 3A and 4C).
12 However, insertions could now also be found at a more terC proximal hotspot
13 region at 3214 kb (yqjI gene) on the chromosome which was150 kb away from
14 the DSB (Figure 4C). This yqjI hotspot was also utilized when DSBs were
15 induced at yhtT-3084::Tn10 which is 290 kb away (Figure 4A). Closer
16 examination of the results indicates that DSBs at multiple positions also induce
17 an oriC proximal hotspot at 3510 kb on the chromosome (the yhfZ gene)(Figures
18 1B and 4B)(Table 4). Taken together, these results demonstrate that there are
19 fixed potential transposition hotspots in the chromosome that can be activated by
20 DSBs over a region of the chromosome that can be hundreds of kilobases from
21 the hotspot (Figures 1 and 4).
22 It appears that closer hotspots are generally used preferentially; for
23 example, when DSBs were induced with yhfT-3084::Tn10, 6 out of 16 insertions
Page 15 DNA damage and Tn7 target activation
1 occurred at the hotspot that was 157 kb away, but only one out of the 16
2 insertions occurred at the hotspot 290 kb away (Figure 4A). However, examining
3 the results found when DSBs were induced at b3219-6::Tn10 suggests that
4 inducing the DSB at a position too close might diminish the attraction to the
5 hotspot; only one of the 14 insertions occurred into a hotspot 19 kb away, but
6 insertions (2/14) could be detected in a hotspot 150 kb away (Figure 4C).
7 Multiple forms of DNA damage induce TnsABC+E transposition:
8 Mitomycin C produces covalent inter-strand cross-links in DNA and can lead to
9 the formation of DSBs. We find that mitomycin C stimulates TnsABC+E
10 transposition, resulting in an average of an additional 103 Lac+ papillae per lawn
11 of cells when compared to growth without mitomycin C (Figure 5A). There was
12 only a small (21 Lac+ papillae per experiment) increase in Lac+ events in the
13 control strain that was likely due to repair related mutagenesis. We also tested
14 the effect of phleomycin on Tn7 transposition which causes a high frequency of
15 DSBs (HUANG et al. 1981; MOORE 1989). TnsE-mediated transposition was
16 stimulated with very low levels of phleomycin that resulted in essentially no
17 increase in the level of background Lac+ events (Figure 5B). We also found a
18 significant increase in TnsABC+E transposition at lower concentrations of
19 phleomycin (2.5 ng/mL). Higher concentrations of phleomycin also stimulated
20 transposition, but cell viability started to be effected (50-100 ng/mL)(data not
21 shown).
22 TnsABC+E transposition was also stimulated when the cells received a
23 brief exposure of short-wave UV light (data not shown)(MATERIALS AND
Page 16 DNA damage and Tn7 target activation
1 METHODS). Exposure to UV light leads to pyrimidine dimers resulting in gaps in
2 nascent daughter-strands that can lead to DSBs (KUZMINOV 2001; RUPP and
3 HOWARD-FLANDERS 1968). UV light exposure was still capable of stimulating
4 transposition even if the exposure did not happen until the lawn of cells was fully
5 grown on the plate, 18 hours into the assay (Figure 5C). Similar to the results
6 found with mitomycin C, some Lac+ cells could be attributed to UV-induced
7 mutagenesis, however, this does not account for the larger increase found with
8 TnsABC+E. For example, for the data shown in Figure 5C, an average of 5
9 additional Lac+ papillae were found in cells without transposition (i.e. Compare
10 TnsABC only strains with and without UV irradiation), as compared to an average
11 of 24 additional Lac+ papillae where TnsE-mediated transposition occurs (i.e.
12 Compare TnsABC+E strains with and without UV irradiation). These results
13 suggest that multiple forms of DNA damage are capable of stimulating
14 TnsABC+E transposition and that the process is not specific to DSBs induced by
15 Tn10 excision.
16 To further investigate the role of DNA damage in stimulating TnsE-
17 mediated transposition we sequenced randomly chosen isolates across 24
18 experiments from strains expressing TnsABC+E that were exposed to UV
19 irradiation (Figure 6)(MATERIALS AND METHODS). TnsE-mediated
20 transposition events occurred at many different regions in the chromosome.
21 Only one of the insertions occurred into a hotspot identified by the direct
22 induction of DSBs (yrbL-mtgA, 3346 kb). Interestingly, we found that insertions
Page 17 DNA damage and Tn7 target activation
1 did seem to occur at two new hotspots, one in zntA (3605-3606 kb) and another
2 between cspA and an IS150 element (3718 kb) (Figure 6).
3 Selection bias does not explain the hotspots in the chromosome:
4 The pattern of TnsE-mediated transposition cannot be simply explained by the
5 activity level of certain Tn7-mediated Lac+ fusions. This would not explain why
6 the profile of insertions changed when DNA breaks were induced at different
7 positions in the chromosome or with UV light (Figures 1, 4, and 6). However, we
8 were interested in knowing if the activity level of the individual Tn7-mediated Lac+
9 fusions found with the papillation assay could explain the hotspots. We
10 determined the β-galactosidase activity of 47 isolates and a negative control,
11 each in triplicate (Figures 6 and S1). These isolates included the samples shown
12 in Figure 6 and other randomly selected isolates from new individual papillation
13 assays. We found that the average β-galactosidase activity level was 261 Miller
14 units, but varied widely from 24 to 1075 with a standard deviation of 262 (Figure
15 S1). The Tn7-directed Lac+ fusions found in the most prominent hotspot 3346
16 (yrbL-mtgA) in Figure 6 was below the average β-galactosidase activity level
17 found among the isolates (Figure 6). As were the insertions that seemed to
18 indicate the two new hotspots at zntR and cspA-IS50 found with UV irradiation
19 (Figure 6). These data support the idea that hotspots do not result from a higher
20 or lower transcription activity of certain target genes. To further ensure that
21 hotspots are not explained by the activity of certain genes we determined the
22 position of the insertions that had a β-galactosidase activity level that was greater
23 than one standard deviation above the average (Figure S1). We found that none
Page 18 DNA damage and Tn7 target activation
1 of these insertions were located in regional hotspots. Four of these insertions
2 were adjacent to or within genes encoding parts of the ribosome (rrlC, yeaD-rrsH,
3 purH-rrsA, and rpmG). The other four insertions were either between or within
4 unrelated genes (yhjD-yhjE, mscL-zntR, farR, and thrC). These results indicate
5 that target gene expression level does not explain the hotspots.
6 TnsE-mediated transposition does not specifically require any one of
7 the known replication restart pathways: The repair of DNA double strand
8 breaks and the repair of DNA damage caused by UV irradiation, mitomycin C,
9 and phleomycin require a mechanism to restart DNA replication forks. Given the
10 results described above, it was possible that TnsE required one of the proteins in
11 the replication restart pathway to recognize target DNAs. The attraction of TnsE-
12 mediated transposition to the terminus region could also be explained by an
13 interaction between this pathway and the replication restart apparatus (BIDNENKO
14 et al. 2006). Replication fork restart can occur through a number of genetically
15 distinct pathways that likely help address the various ways a DNA replication fork
16 can stall or collapse (HELLER and MARIANS 2006). We determined if any of the
17 multiple pathways for replication fork restart was specifically required for TnsE-
18 mediated transposition during normal growth.
19 PriA is a 3’ to 5’ helicase that is required for the major replication fork
20 restart pathways in E. coli (SANDLER and MARIANS 2000). We monitored
21 transposition in a ∆priA strain and found that this component of the restart
22 apparatus was not required for TnsE-mediated transposition (Figure 7).
23 Surprisingly, we found that TnsE-mediated transposition was actually stimulated
Page 19 DNA damage and Tn7 target activation
1 in ∆priA strains. This effect was specific to TnsE-mediated transposition because
2 no change was found in TnsABC* transposition; the TnsABC* core machinery
3 has a mutant TnsCA225V that does not require TnsE for targeting or activation
4 (STELLWAGEN and CRAIG 1997b). It is possible that the stimulatory effect found in
5 the ∆priA background might be caused by TnsE favoring a structure or complex
6 that is processed more slowly in a ∆priA background. To further rule out any
7 requirement for PriA, we utilized a gain of function mutation in dnaC, called
8 dnaC809, that allows replication restart in a ∆priA background. DnaC loads the
9 replicative helicase, DnaB, as part of initiating all DNA replication forks
10 (KORNBERG and BAKER 1992). The dnaC809 allele bypasses the need for
11 multiple other replication fork restart proteins like PriB and DnaT, in vivo and in
12 vitro (LIU et al. 1999; SANDLER et al. 1996). TnsABC+E transposition was
13 unaffected by the dnaC809 allele (Figure 7). The results found with the ∆priA
14 and dnaC809 strains indicates that the replication fork restart pathway that
15 requires the PriA, PriB and DnaT proteins is not required for TnsE transposition
16 (i.e. a specific interaction between TnsE and one of these proteins or a specific
17 structure made by these proteins is not required for transposition in vivo).
18 PriA is essential for the major replication fork restart pathways involving
19 PriB and a second pathway involving PriC (SANDLER 2005). However, PriC can
20 also restart DNA replication forks in a ∆priA background through the use of a
21 second DNA 3’ to 5’ DNA helicase, called Rep. We monitored transposition in a
22 ∆priC strain to determine if the PriC-Rep pathway of replication fork restart was
23 required for TnsE-mediated transposition (Figure 8). We find that TnsABC+E
Page 20 DNA damage and Tn7 target activation
1 transposition is unaffected by the priC allele when compared with the TnsABC*
2 control strain. Taken together, the observation that PriA and PriC are not
3 required for transposition indicates that no protein that is required for these
4 pathways is an essential host factor for TnsE-mediated transposition. This
5 includes the known pathways that require PriB, DnaT, and Rep. It is unknown
6 why TnsABC+E transposition is stimulated in ∆priA strains, but this could indicate
7 that slowed DNA repair allows a structure or complex that is preferred by TnsE to
8 linger in the cell. While a priA suppressor mutation is not sufficient to stimulate
9 TnsE-mediated transposition (Figure 7), it remains possible that suppressor
10 mutations in dnaC can facilitate a priA-independent process preferred by TnsE-
11 mediated transposition.
12 Induction of the SOS response does not stimulate TnsE-mediated
13 transposition: The many types of DNA damage that stimulate TnsE-mediated
14 transposition also stimulate the SOS response, thereby leaving the possibility
15 that induction of the SOS response might actually be responsible for the increase
16 in TnsE-mediated transposition. To test this idea we monitored transposition in a
17 strain background that did not have the LexA protein that is responsible for
18 repressing the genes otherwise expressed in the SOS response (MATERIALS
19 AND METHODS). To accurately gauge the effect of the SOS response we also
20 included a sulA allele; induction of SulA during the SOS response results in the
21 formation of cell filaments. We find that constitutive SOS induction in the lexA
22 backgrounds does not stimulate TnsE-mediated transposition (Figure 9). Instead
23 we actually find a significant drop in all transposition in the sulA lexA background,
Page 21 DNA damage and Tn7 target activation
1 which may be explained by one or more of the genes in the SOS regulon
2 effecting cell viability.
3
4
5 DISCUSSION
6 When DSBs are induced at specific positions in the E. coli chromosome,
7 TnsE-mediated transposition is stimulated to occur preferentially at one or more
8 discrete hotspots (Figures 1, 3, and 4). We show that these hotspots are fixed in
9 the chromosome and are only activated when the DSB is induced in the same
10 region of the chromosome (Figure 4)(Table 4). We find that multiple forms of
11 DNA damage stimulate TnsABC+E transposition (Figures 5 and 6), however, this
12 is not explained simply by induction of the SOS response (Figure 9). Although
13 replication fork restart is required after DNA damage, no single pathway of
14 replication fork restart nor RecBCD is an essential target for TnsE-meditated
15 transposition (Figures 2, 7 and 8). Our results are consistent with the idea that
16 the replication-mediated repair of DSBs activates regional loci in the
17 chromosome that can be targeted by TnsABC+E transposition.
18 Replication involved in repair may be recognized by TnsABC+E
19 transposition: Given that TnsABC+E transposition recognizes an aspect of DNA
20 replication in conjugal plasmids and the chromosome (PETERS and CRAIG 2001a),
21 it seems reasonable to speculate that replication involved in DNA repair could
22 also be a target for TnsABC+E transposition. This idea is supported by the
23 distribution of TnsE-mediated insertions that are stimulated by DSBs in three
Page 22 DNA damage and Tn7 target activation
1 ways: First, in almost every case, the left to right orientation of the ends of the
2 insertions at hotspots are consistent with an orientation predicted if the
3 replication fork started at the DSB (PETERS and CRAIG 2001a). For example,
4 transposition events in hotspots at aroG(786 kb), lysR(2976-2977 kb), mscL-
5 rplQ(3436-3437), and yhfZ(3510 kb) that were all on the oriC proximal side of the
6 induced DSB were in the opposite orientation of the insertions in the terC
7 proximal hotspots at serW(925 kb), yqiJ(3214kb), yrb-mtgA(3346-3347 kb), and
8 yhcC-gltB(3352 kb)(Figures 1 and 4)(PETERS and CRAIG 2000). Second, most of
9 the hotspots activated by induced DSBs are proximal to the terminus and not the
10 origin of DNA replication (Figures 1 and 4). The distribution of actively oriented
11 chi sites makes it much more likely that replication forks will be shunted towards
12 the terminus (KOWALCZYKOWSKI 2000; KUZMINOV 1999). The most obvious
13 hotspot that was on the oriC proximal side of the DSB was found in the recD-
14 1901::Tn10 strain, a strain where chi sites will not be recognized by the
15 RecBC+(D-) enzyme thereby allowing replication forks to progress in either
16 direction from the DSB (AMUNDSEN et al. 1986; CHAUDHURY and SMITH 1984).
17 Third, the one hotspot that appeared to be stimulated by DSBs at the farthest
18 distributed locations (zbi-29::Tn10, b3219-6::Tn10, or yhf-3084::Tn10) is only
19 10kb from terC (Figures 1D, 3B, 4A, and 4C). Given that replication forks
20 initiated at DSBs throughout the chromosome would be directed to terC, this
21 result is also consistent with the idea that hotspots are activated by DNA
22 replication from DNA repair. DSBs are also associated with UV irradiation,
23 exposure to mitomycin C and phleomycin and could provide a unifying target
Page 23 DNA damage and Tn7 target activation
1 across these agents of DNA damage for TnsABC+E transposition (Figure 5). It is
2 especially significant that phleomycin had the greatest stimulatory effect on
3 transposition given that this drug is associated with the direct formation of DSBs
4 (HUANG et al. 1981; MOORE 1989).
5 What causes the transposition hotspots? Even if DNA replication
6 associated with DNA repair is being targeted by TnsE-mediated transposition it
7 would not immediately explain the hotspots found in the chromosome. It is
8 possible that the hotspots are positions where forks initiated for DSB repair stall
9 in the chromosome, much in the same way that normal DNA replication appears
10 to be a preferred target when replication terminates proximal to terC (Figure
11 10A). There are multiple intriguing examples where hotspots are found where
12 replication forks might be predicted to stall. Replication forks have been found to
13 stall at tRNA genes and other highly expressed genes when transcription
14 complexes may collide “head-on” with DNA replication forks (DESHPANDE and
15 NEWLON 1996; MIRKIN and MIRKIN 2007). This could indicate that the serW tRNA
16 gene acts like a hotspot because of replication fork stalling. Of some interest, the
17 serW locus may be especially sensitive to recombination given that the serW
18 tRNA gene is also a known position for the occurrence of pathogenicity islands
19 (TAYLOR et al. 2002). The hotspot at 3436-3437 (mscL-rplQ) is at the end of a
20 large operon that includes many highly transcribed essential genes including
21 ribosomal subunits, the alpha subunit of RNA polymerase, and secY. This
22 hotspot was only activated when DSBs were induced in a region predicted to
23 send DNA replication forks in a head on collision with transcription complexes.
Page 24 DNA damage and Tn7 target activation
1 If the hotspots are positions where replication forks can stall, this process
2 could be compounded over time during the assay. This is because DSBs are
3 continually induced during the multiple day assays that would presumably allow
4 replication forks to “catch up” to previous forks at the stall site (Figure 10A). If
5 the DSB was sufficiently close to a replication fork stall site it is possible that the
6 RecBCD enzyme could degrade the non-contiguous arm of the stalled replication
7 fork (i.e. at the DSB, the oriC-proximal broken end is likely used to initiate DNA
8 replication on a sister chromosome, but the terC-proximal broken end must be
9 degraded)(Figure 10B). This could provide an explanation for why hotspots are
10 strongly activated by DSBs 157 and 83 kb from the hotspot, but not when DSBs
11 are induced 19 kb from the hotspot (Figure 4); at larger distances there may be
12 insufficient time for processing of the broken end before arrival of the fork
13 initiated at the DSB (Figure 10B). While the RecBCD enzyme is very processive,
14 it will dissociate after ~30 kb. This model is also supported by the observation
15 that significant oriC proximal hotspots are only found in the recD strain; The
16 RecBC(D-) enzyme does not require chi sites to load RecA.
17 Our results with the ∆priA allele could also support a model where
18 hotspots result from stalled replication forks. Repair pathways involving PriA or
19 PriC must restart DNA replication when forks stall or collapse at DNA damage
20 (HELLER and MARIANS 2005). While neither pathway is specifically required for
21 TnsABC+E transposition, TnsABC+E transposition is sensitive to which pathway
22 is used (Figures 7 and 8). Strains with the ∆priA allele must use the PriC
23 pathway, which may be less efficient at restarting replication on certain kinds of
Page 25 DNA damage and Tn7 target activation
1 substrates, possibly resulting in persistently stalled replication forks somewhere
2 in the chromosome (HELLER and MARIANS 2005).
3 It is important to note that many of the hotspots occur in regions where
4 head on collisions between replication and transcription complexes are not
5 expected. It is possible that replication forks could stall by other mechanisms at
6 these positions. While many of the hotspots are in genes of unknown function,
7 there is no obvious commonality in function between hotspots found in well-
8 studied genes (data not shown)(Table 4). We also find no obvious consensus
9 sequence between the multiple regional hotspots (data not shown).
10 We find that neither the hotspots nor the differential activation of hotspots
11 results from the expression level of genes in the chromosome. Of 47 randomly
12 selected Tn7-based Lac+ fusions that were examined, none of the most
13 transcriptionally active isolates resulted from insertions into hotspots (Figures 6
14 and S1). The insertions into hotspots generally produced below average levels
15 of β-galactosidase activity (Figure 6). Therefore, while there are limitations to the
16 promoter-based assay used in this study, we clearly observe a phenomenon that
17 is specific to the TnsE-mediated pathway of Tn7 transposition following DNA
18 damage.
19 Hotspots reflect a finer organization feature than any known
20 macrodomain structure of the chromosome: Various types of barriers have
21 been demonstrated in the E. coli chromosome using a variety of genetic and
22 physical assays. Experiments suggest that the chromosome is organized into six
23 large macrodomains with largely fixed barriers based on the ability of site-specific
Page 26 DNA damage and Tn7 target activation
1 recombination sequences to interact (VALENS et al. 2004). The Tn7 hotspots do
2 not coincide with the barriers of these macrodomains suggesting hotspots do not
3 result from this domain structure. While the transposition hotspots usually seem
4 to be in the same macrodomain as the induced DNA double strand break, this is
5 likely explained by the large size of the macrodomains and that the genetic
6 position of the macrodomain boundaries remains loosely defined. Other
7 experiments have indicated that fluid barriers in the chromosome appear to exist
8 that constrain the chromosome in units of around 10 kb (DENG et al. 2004;
9 POSTOW et al. 2004). However, we find transposition hotspots with a wide
10 distribution of the insertions. It is intriguing that another transposon, IS903, has a
11 preference for small regional hotspots (SWINGLE et al. 2004). However, any
12 mechanistic connection between IS903 and Tn7 is unclear; there is no overlap
13 with the hotspots used with these elements and the IS903 hotspots do not need
14 to be activated by regional DSBs.
15 Future experiments will focus on determining the precise nature of the
16 transposition hotspots activated by DNA damage. These studies may illuminate
17 biological adaptations for genome stability.
18
Page 27 DNA damage and Tn7 target activation
1 TABLE 1
2 Strains used in this study
Strain Genotype Reference
MC4100 araD139 ∆(argF-lac)169 rpsL150 relA1 (CASADABAN
flhD5301 deoC1 ptsF25 rbsR22 e14- ∆(fimB- 1976; PETERS et
fimE)632::IS1 ∆(fruK-yeiR)725 al. 2003)
R NLC28 MC4100 Val (MCKOWN et al.
1987)
R JP617 NLC28 attTn7::miniTn7(R90-lacZYA’-Kan - (MCKOWN et al.
L166) 1988; PETERS and
CRAIG 2000)
JP1588 JP617 priA::CamR Nancy Craig
AP44 JP617 dnaC809 P1(JC19008) X
AP40
AP48 JP617 dnaC809 priA::CamR P1(JP1588) X
AP44
Page 28 DNA damage and Tn7 target activation
JP1386 NLC28 ∆ara714 (PETERS and
CRAIG 2001a)
AP429 JP1386 attTn7::miniTn7(R90-lacZYA’-KanR- P1(JP617) X
L166) JP1386
AP453 JP1386 attTn7::miniTn7(R90-lacZYA’-frt-CamR- This work
frt-L166)
AP457 JP1386 attTn7::miniTn7(R90-lacZYA’-frt-L166) This work
AP523 AP457 priC303::KanR P1(JC19008) X
AP457
AP767 AP457 ∆sulA6209::TetR P1(GM7542) X
AP457
AP964 AP457 lexA71::Tn5 P1(KL788) X
AP457
AP965 AP457 lexA71::Tn5 ∆sulA6209::TetR P1(KL788) X
AP767
AP40 JP617 thrA-34::Tn10 P1(CAG18442) X
JP617
JP949 NLC28 attTn7::Tn7 Nancy Craig
AP699 BW25113 attTn7::CamR This work
Page 29 DNA damage and Tn7 target activation
AP724 NLC28 attTn7::CamR This work
JP2178 NLC28 attTn7::miniTn7(R90-lacZYA’-frt-L166) This work
srlD::Tn10 ∆sbcDC::dhfr
BW25113 MG1655 ∆(araD-araB)567 lacZ-4787∆(::rrnB-3) (DATSENKO and
∆(rhaD-rhaB)568 hsdR514 WANNER 2000)
CAG12135 MG1655 recD-1901::Tn10 (NICHOLS et al.
1998; SINGER et
al. 1989)
CAG12153 MG1655 b3219-6::Tn10 (NICHOLS et al.
1998; SINGER et
al. 1989)
CAG12071 MG1655 smg-3082::Tn10 (NICHOLS et al.
1998; SINGER et
al. 1989)
CAG18433 MG1655 zbf-3057::Tn10 (NICHOLS et al.
1998; SINGER et
al. 1989)
CAG18442 MG1655 thrA-34::Tn10 (NICHOLS et al.
1998; SINGER et
al. 1989)
Page 30 DNA damage and Tn7 target activation
CAG18456 MG1655 yhfT-3084::Tn10 (NICHOLS et al.
1998; SINGER et
al. 1989)
CAG18493 MG1655 zbi-29::Tn10 (NICHOLS et al.
1998; SINGER et
al. 1989)
CAG18642 MG1655 srlD-3131::Tn10 (NICHOLS et al.
1998; SINGER et
al. 1989)
GM7542 MG1655 ∆sulA6209::TetR Martin Marinus
via Mark Sutton
+ R JC19008 JC18983 dnaC809 thrA priC303::Kan (SANDLER et al.
1996)
KL788 ∆(gpt-lac)5 tsx-35 sulA3 e14- rfbC1 recA441(ts) (MILLER and LOW
relA1 rpsL31 kdgK51 mtl-1 spoT1 thi-1 1984)
lexA71::Tn5 creC510
STL7206 MG1655 sbcB::frt-KanR-frt rph-1 Susan Lovett
STL7886 MG1655 recC::frt-CamR-frt rph-1 Susan Lovett
1
2
Page 31 DNA damage and Tn7 target activation
1 TABLE 2
2 Plasmids used in this study and the source or construction
Name Relevant information
pCW4 pACYC184 derivative encoding TnsABCDE, tetracycline resistant
(WADDELL and CRAIG 1988).
pCW15 pACYC184 derivative encoding TnsABC, chloramphenicol resistant
(WADDELL and CRAIG 1988).
pCW15* pACYC184 derivative encoding TnsABCA225V, chloramphenicol
resistant (STELLWAGEN and CRAIG 1997b).
pJP123 pACYC184 derivative encoding TnsABC+E, chloramphenicol
resistant (PETERS and CRAIG 2000)
pTA106 pSC101 replicon, ampicillin resistant cloning vector (PETERS and
CRAIG 2000).
pJP104 pTA106 derivative encoding TnsE, ampicillin resistant (PETERS and
CRAIG 2000).
pQS100 pTA106 vector encoding TnsABC constructed by cloning the 4919 bp
of PvuII fragment encoding TnsABC from pCW4 into the SmaI site of
pTA106 with the tnsC gene proximal to the vector HindIII site
pQS101 pTA106 vector encoding TnsABC constructed by cloning the 4919 bp
of PvuII fragment encoding TnsABC from pCW4 into the SmaI site of
pTA106 with the tnsA gene proximal to the vector HindIII site
pQS102 pTA106 vector encoding TnsABC+E constructed by cloning the 4919
bp of PvuII fragment encoding TnsABC from pCW4 into the SmaI
Page 32 DNA damage and Tn7 target activation
site of pJP104 vector with the tnsC gene proximal to the vector
HindIII site
pQS103 pTA106 vector encoding TnsABC+E constructed by cloning the 4919
bp PvuII fragment encoding TnsABC from pCW4 into the SmaI site
of pJP104 vector with the tnsA gene proximal to the vector HindIII
site.
pQS107 pTA106 vector encoding TnsABCA225V constructed by cloning a 726
bp KpnI-StuI fragment encoding the Alanine(225) to Valine amino
acid change of tnsC from pCW15* into pQS101.
pZT383 pMB1 vector with lacIq regulation expressing Tn10 transposase
mutant P167S, ampicillin resistant (GALITSKI and ROTH 1997)
pJP135 pMB1 vector with lacIq regulation expressing Tn10 transposase
mutant D97A/D161A, ampicillin resistant (PETERS and CRAIG 2000)
pCP20 Temperature sensitive plasmid with thermal induction of FLP
recombinase (CHEREPANOV and WACKERNAGEL 1995).
pKD46 Temperature sensitive plasmid with the λ Red proteins under
arabinose control (DATSENKO and WANNER 2000).
pKD3 Plasmid encoding ampicillin resistance that allows PCR amplification
of a gene cassette encoding chloramphenicol resistance flanked by
frt sites recognized by the FLP recombinase (DATSENKO and WANNER
2000).
1
2
Page 33 DNA damage and Tn7 target activation
1
2
3 TABLE 3
4 DSBs stimulate TnsE-mediated transposition.
5
Tn10 No DSBs Yes DSBs Fold increase
insertion TnsABC TnsABC TnsABC TnsABC TnsABC+E
allele +E +E transposition above
background
zbi-29 17a (1.6)b 11 (1.1) 22 (1.5) 35 (7.3) 3.2
recD-1901 12 (1.5) 28 (1.6) 22 (1.0) 71 (4.7) 2.5
b3219-6 7 (0.8) 12 (1.6) 16 (2.0) 44 (4.0) 3.7
smg-3082 1 (0.2) 1 (0.2) 2 (0.7) 6 (1.0) 6.0
yhfT-3084c 11 (0.7) 17 (2.7) 22 (1.8) 52 (4.7) 3.1
6 a Average number of papillae from 12 experiments.
7 b Standard error of the mean.
8 c Data from the experiment shown in Figure 4A
9
Page 34 DNA damage and Tn7 target activation
1 TABLE 4
2 Transposition hotspots and chi site frequency around DSBs.
Tn10 Total Tn7 chi sites before the insertion / kb before the insertion insertion insertions insertions First Second Third Fourth Fifth Sixth Seventh
allele a examined a on the Insertion Insertion Insertion Insertion Insertion Insertion Insertion
(Position) (Percent oriC or
within terC side
300kb of of the
the DSB) break
zbf-3057 7 (57%) oriC 8 / 74 * / * * / * * / * * / * * / * * / *
(698 kb) terC 6 / 55 * / 227b * / 227b * / * * / * * / * * / *
zbi-29 13 (46%) oriC 3 / 33 3 / 34c 3 / 34c * / * * / * * / * * / *
(820 kb) terC 14 / 106b 20 / 164 * / 186 * / * * / * * / * * / * recD-1901 15 (73%) oriC 8 / 27d 8 / 27d 8 / 28d 8 / 28d 19 / 106 19/106 */289
(2949 kb) terC 10 / 122 10 / 146 10 / 154 10 / 227e * / * * / * * / * srlD-3131 10 (70%) oriC 6 / 37 * / 150 * / * * / * * / * * / * * / *
(2826 kb) terC 0/10 0/74 0/104e 0/104e 3/143 * / * * / *
b3219-6 14 (50%) oriC 8 / 61 11 / 71f * / * * / * * / * * / * * / *
(3364 kb) terC 0 / 19g 5 / 126 8 / 150h 8 / 150h 11 / * * / * * / * smg-3082 11(82%) oriC 0 / 7f 11 / 80i * / * * / * * / * * / * * / *
(3430 kb) terC 3 / 78j 3/ 83g 3/ 83g 3/ 83g 3/ 83g 3/ 83g 3/ 84g yhfT-3084 28 (54%)k oriC 2 / 6i 7 / 40 * / * * / * * / * * / * * / *
(3504 kb) terC 4 / 152j 4 / 157g 4 / 157g 4 / 157g 4 / 157g 4 / 157g 4 / 157g 3 a Data for the zbf-3057 and srlD-3131 alleles were from the published insertions
4 (PETERS and CRAIG 2000). The remaining data was from the following: zbi-29
5 (Fig.1D), recD-1901 (Figure 1C), b3219-6 (Figure 4C), smg-3082 (Figure 4B),
6 and yhfT-3084 (Figure 1A and 4A).
7 b Hotspot at 925 kb (serW), three insertions 346 bp apart
8 c Hotspot at 786 kb (aroG), two insertions 69 bp apart
9 d Hotspot at 2976-2977 kb (lysR), four insertions 842 bp apart
Page 35 DNA damage and Tn7 target activation
1 e Hotspot at 2722-2723 kb (yfiM and kgtP), three insertions 333 bp apart
2 f Hotspot at 3426-3437 (mscL-rplQ), two insertions 1074 bp apart
3 g Hotspot at 3346-3347 kb (mtgA and yrbL), seventeen insertion 820 bp apart
4 h Hotspot at 3214 kb (yqiJ), three insertions 282 bp apart (includes insertion
5 induced by the yhfT-3084::Tn10)
6 i Hotspot at 3510 kb (yhfZ), two insertions 14 bp apart
7 j Hotspot at 3352 kb (yhcC-gltB), two insertions 420 bp apart
8 k An additional 8 insertions were within 300kb because of the larger number of
9 samples, but the percentage within 300kb remained the same.
10 * Indicates over 20 chi sites or over 300kb
11
Page 36 DNA damage and Tn7 target activation
1 Figure captions
2 FIGURE 1 – Inducing DNA DSBs specifically stimulates TnsABC+E transposition
3 events that occur proximal to the DSB site with a preference for hotspots.
4 Transposition was monitored with a promoter capture assay using strain JP617
5 derivatives on MacConkey’s media (MATERIALS AND METHODS). The graph
6 (A) shows the frequency of transposition indicated by the number of Lac+ papillae
7 after 72 hours at 30˚ when DSBs were induced with yhfT-3084::Tn10 on
8 MacConkey’s media. Error bars indicate the standard error of the mean
9 (n=12)(data from other positions are shown in Table 3). Circles represent the E.
10 coli chromosome. Individual insertions from separate experiments using strains
11 expressing the TnsABC+E proteins where DSBs were induced are indicated with
12 arrows. Arrows outside the circle indicate right to left insertions, arrows inside
13 the circle indicate left to right insertions. The number indicates the position of the
14 insertion on the E. coli genome in kb. Insertions that occurred into one of the
15 hotspots found in this work are indicated with an asterisk (*). The gene where
16 the Tn10-generated DSB was induced is indicated in a box (and its position in
17 kilo base pairs). oriC and its position in kb are marked. Triangles indicate the
18 position of the ten ter sites in the chromosome, terC is indicated with its position
19 in kb. The JP617 background strains were used to induce DSBs using a Tn10
20 insertion at one of three positions; yhfT (yhfT-3084::Tn10 from CAG18456) (B),
21 recD (recD-1901::Tn10 from CAG12135) (C), or zbi (zbi-29::Tn10 from
22 CAG18493)(D). Breaks were induced with pZT383 (Yes-Breaks) or the strain
23 had a control plasmid pJP135 (No – Breaks). Tn7 transposition functions were
Page 37 DNA damage and Tn7 target activation
1 expressed from pJP123 (TnsABC+E) or a vector that does not allow
2 transposition was used, pCW15 (TnsABC).
3
4 FIGURE 2 – Double-strand DNA breaks stimulate TnsE-mediated transposition
5 in the absence of RecBCD. Transposition was monitored with a promoter capture
6 assay using strain JP617 derivatives on LB media with lactose and X-Gal when
7 DSBs were induced at srlD3131::Tn10(MATERIALS AND METHODS)(PETERS
8 and CRAIG 2000). Breaks were induced with pZT383 (Yes-Breaks) or the strain
9 had a control plasmid pJP135 (No – Breaks). Tn7 transposition functions were
10 expressed from pJP123 (TnsABC+E) or a vector that does not allow
11 transposition was used, pCW15 (TnsABC).The graph shows the frequency of
12 transposition indicated by the number of Lac+ papillae after 120 hours at 30˚ from
13 12 independent experiments. Error bars indicate the standard error of the mean.
14
15 FIGURE 3 –DSBs stimulate TnsABC+E transposition at multiple different
16 positions in the yrbL-mtgA and marR/ydeE-ydeH hotspots. Filled arrows indicate
17 the relative position of TnsE-mediated insertion events, filled arrows above the
18 line indicate left to right insertions, filled arrows below the line indicate right to left
19 insertions. (A) The yrbL-mtgA hotspot (3346-3347 kb). Insertions were from
20 Figures 1B ,4A, 4B, 4C. (B) The marR/ydeE-ydeH hotspots (1617/1620-1621).
21 Insertions were from Figure 1D, 4A, and 4C. The number indicates the Tn10
22 allele that was used to induce DSBs in the strain; yhfT-3084::Tn10 (1), smg-
23 3082::Tn10 (2), and b3219-6::Tn10 (3), and zbi-29::Tn10 (4). Unfilled arrows
Page 38 DNA damage and Tn7 target activation
1 indicate the orientation of the open reading frames. Predicted sigma 70
2 promoters are shown with bent arrows and indicated (P) from PEC
3 (http://www.shigen.nig.ac.jp/ecoli/pec/). The oriC and terC proximal sides of the
4 region are shown.
5
6 FIGURE 4 – Inducing DSBs specifically stimulates TnsABC+E transposition
7 events that occur proximal to the DSB site with a preference for fixed regional
8 hotspots. Transposition was monitored with a promoter capture assay using
9 strain JP617 derivatives on MacConkey’s media (MATERIALS AND METHODS).
10 The position of transposition events from strains with the TnsABC+E proteins
11 where DSBs were induced are shown. Designations are as in Figure 1. The
12 three relevant loci, yhfT, smg, and b3219, are indicated (and the position shown
13 in kb). The position where the DSB was induced is indicated in bold and boxed.
14 The JP617 background strains were used to induce DSBs using a Tn10 insertion
15 at one of three positions; yhfT (yhfT-3084::Tn10 from CAG18456) (A), smg (smg-
16 3082::Tn10 from CAG12071) (B), or b3219 (b3219-6::Tn10 from CAG12153)(C).
17 Breaks were induced with pZT383 (Yes-Breaks) or the strain had a control
18 plasmid pJP135 (No – Breaks). Tn7 transposition functions were expressed from
19 pJP123 (TnsABC+E) or a vector that does not allow transposition was used,
20 pCW15 (TnsABC).The locations of two relevant hotspots are indicated with small
21 circles, yqiJ (3214 kb) and yrbL-mtgA (3346-3347 kb).
22
Page 39 DNA damage and Tn7 target activation
1 FIGURE 5 –Mitomycin C, phleomycin, and UV exposure stimulate TnsABC+E
2 transposition. Tn7 functions were expressed from pQS101 (TnsABC) or pQS103
3 (TnsABC+ TnsE). Transposition was monitored with a promoter capture assay
4 using strain JP617 on MacConkey’s media (MATERIALS AND METHODS). (A)
5 The frequency of transposition is indicated on the Y axis as the number of Lac+
6 papillae after 72 hours at 30˚ on MacConkey’s plates with and without mitomycin
7 C. (B) The frequency of transposition is indicated on the Y axis as the number of
8 Lac+ papillae after 72 hours at 30˚ on MacConkey’s plates with and without
9 phleomycin. (C) After 18 hours of incubation at 30˚C one set of plates was
10 exposed to 10 millijoules of UV light (Yes UV) and the other was left unexposed
11 as a control (No UV). The frequency of transposition is indicated on the Y-axis as
12 the number of Lac+ papillae after 72 hours at 30˚C post-UV light on MacConkey’s
13 plates. Error bars indicate the standard error of the mean (n=12).
14
15 FIGURE 6 – TnsE-mediated transposition events were stimulated to occur at
16 many locations in the chromosome after exposure to UV irradiation. 24
17 transposition randomly chosen events were mapped and sequences from the
18 Lac+ papillae found in Figure 5B and a replicate of this experiment. Designations
19 are as in Figure 1. Next to each individual insertion the average β-galactosidase
20 activity of the resulting fusion is shown (n=3) with the standard deviation of the
21 mean (See text for details).
22
Page 40 DNA damage and Tn7 target activation
1 FIGURE 7 - The PriA protein is not required for TnsABC+E transposition. Tn7
2 functions were expressed from pQS100 (TnsABC), pQS107 (TnsABC*), or
3 pQS102 (TnsABC+ TnsE). The TnsABC* mutant core machinery that allows
4 untargeted transposition is included as a positive control to rule out effects that
5 are not associated with TnsE targeted transposition (✶). Transposition was
6 monitored with a promoter capture assay using strains JP617 (WT), JP1588
7 (priA), AP44 (dnaC809), and AP48 (dnaC809 priA)(MATERIALS AND
8 METHODS). The frequency of transposition is indicated on the Y-axis as the
9 number of Lac+ papillae after 72 hours at 30˚C on MacConkey’s media. Error
10 bars indicate the standard error of the mean (n=12).
11
12 FIGURE 8 - The PriC protein is not required for TnsABC+E transposition. Tn7
13 functions were expressed from pQS100 (TnsABC), pQS102 (TnsABC+ TnsE), or
14 pQS107 (TnsABC*). The TnsABC* mutant core machinery that allows untargeted
15 transposition is included as a positive control to rule out effects that are not
16 associated with TnsE targeted transposition (✶). Transposition was monitored
17 with a promoter capture assay using strains AP457 (WT) and AP523
18 (priC)(MATERIALS AND METHODS). The frequency of transposition is indicated
19 on the Y-axis as the number of Lac+ papillae after 72 hours at 30˚ on
20 MacConkey’s media. Error bars indicate the standard error of the mean (n=12).
21
22 FIGURE 9 – Induction of the SOS response does not stimulate TnsABC+E
23 transposition. Tn7 functions were expressed from pQS100 (TnsABC), pQS102
Page 41 DNA damage and Tn7 target activation
1 (TnsABC+ TnsE), or pQS107 (TnsABC*). The TnsABC* mutant core machinery
2 that allows untargeted transposition is included as a positive control to rule out
3 effects that are not associated with TnsE targeted transposition (✶).
4 Transposition was monitored with a promoter capture assay using strains AP457
5 (WT), AP767 (sulA), AP964 (lexA) and AP965 (sulA lexA)(MATERIALS AND
6 METHODS). The frequency of transposition is indicated on the Y-axis as the
7 number of Lac+ papillae after 72 hours at 30˚ on MacConkey’s media. Error bars
8 indicate the standard error of the mean (n=12).
9
10 FIGURE 10 - A model explaining how DNA replication forks initiated for DSB
11 repair could lead to hotspots where DNA replication forks stall. Additionally the
12 model explains why sites where replication forks stall that are close to a DSB
13 may not form hotspots, but sites further from the DSB could form hotspots. Both
14 panels show a chromosome that has been partially replicated from the single
15 origin of DNA replication. Panel “A” indicates a hypothetical situation where a fork
16 stall site is far from a DSB and panel “B” indicates a stall site close to a DSB. In
17 both cases the RecBCD enzyme should degrade at both ends of the DSB.
18 However, the RecBCD enzyme that processes the DSB end towards the origin is
19 quickly converted to a replication fork progressing towards the terminus because
20 of frequent chi sequences in one orientation. The RecBCD enzyme is indicated
21 in cross-hatching and the site where replication forks stall is indicated with a
22 small circle. See text for details.
23
24
Page 42 DNA damage and Tn7 target activation
1 ACKNOWLEDGMENTS
2 We thank Nancy Craig (Johns Hopkins Medical School, Howard Hughes
3 Medical Institute), Susan Lovett (Brandeis University), Steve Sandler (University
4 of Massachusetts at Amherst), Mark Sutton (University at Buffalo), and the E. coli
5 Genetic Stock Center, Yale University for providing strains and plasmids. We
6 thank the anonymous reviewers for suggesting useful experiments, Zaoping Li
7 for comments on the manuscript, and David Milller and Brian Kvitko for
8 preliminary work as rotation students. This work was funded by a grant from the
9 National Science Foundation (MCB-0315316).
10
Page 43 DNA damage and Tn7 target activation
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Page 50 DNA damage and Tn7 target activation
1
2
Page 51 Fig.1
A B 60
r
e TnsABC TnsABC+E 50 oriC(3923)
b 134
m 40
u
n 30 3510* e 3352*
a
l 3347* l 20
i
p yhfT(3504) 3347* a 10 3347*
P 3346* terC(1607) 0 1234 3250 1432 No Yes No Yes 2771 1551 Breaks
787 C D zbi(820) 786* 4058 786* 925* 984 1006 3238 1488 3055 1509 1617* 3057 2827 1621* 2803 2977* 1551 1621* 2977* 2795 1988 2976* 1639 2976* recD(2949) 2722 2173 2454 Fig.2
50 Wild type recC recC 45 sbcBCD 40 35 30 25 20
Papillae number Papillae 15 10 5 0 Breaks No123456789101112 Yes No Yes No Yes No Yes No Yes No Yes Tns ABC ABC+E ABC ABC+E ABC ABC+E Fig.3
1 11 1 3 1 1 1 2 2 2 1 P A 2 2 1 1 P P 2 P terC oriC ptsO yrbL mtgA yhbL
3 4 4 B P P P 4 P P terC oriC marC marA ydeD ydeE ydeH 3 marR marB 1 233 Fig.4 A
oriC(3923) 4614
3934 1018 3543 3347* yhfT(3504) 3347* 3347* smg(3430) 3346* 3346* b3219(3364) 3346* terC(1607) 3214* 3040 2945 2532 1620*
2494 B 4404
3510* yhfT(3504) 3352* 3437* 3347* smg(3430) 3347* 3347* 3346* b3219(3364) 3346* 3346* 2809
C 4241 0.288
yhfT(3504) 3638 3436* smg(3430) 1381 3426 3346* 3238 b3219(3364) 3214* 3214* 1617* 3054 1621*
2725 Fig.5
A 160 BC120 80 No Mitomycin C Mitomycin C No Phleomycin Phleomycin No UV Yes UV 140 100 ng/mL 10 ng/mL 70
r
r
r 100
e
e
e
b
b b 120 60
m
m
m
80 u
u u 100
n
n
n 50
e
e
e
a
a
a
l 80 l 60
l l 40
l
l
i
i
i
p
p
p
a
a a 60 30
P P 40
P 40 20 20 20 10
0 1234 0 1234 0 1234 TnsABC + + + + TnsABC + + + + TnsABC + + + + TnsE - + - + TnsE - + - + TnsE - + - + Fig.6
135 (29+/-1) 4242 (126+/-12) 233 (92+/-3) 4240 (124+/-6) 4619 (166+/-2)
4188 (170+/-2) 4371 (105+/-1) 4248 (99+/-2) 4246 (88+/-1) 3909 (81+/-2) 3672 (891+/-2) 3718 (238+/-7) 3718 (202+/-5) 3606 (129+/-4) 3606 (192+/-1) 3436 (174+/-3) 3605 (184+/-2)
3346* (135+/-7) 3276 (232+/-31)
3231 (207+/-8) 1489 (116+/-4) 2755 (144+/-1) 2042 (64+/-4) 1609 (67+/-7) Fig.7
300 WT priA dnaC809 priA dnaC809 250
r
e
b 200
m
u
n 150
e
a
l
l
i
p
a 100
P
50
0 TnsABC +123456789101112 ++ + + + + + TnsE - - + - - + - - + - - + Fig.8
200 180 WT priC 160
r
e 140
b
m 120
u
n
e 100
a
l
l i 80
p
a
P 60 40 20 0 TnsABC +123456 ++ + TnsE - - + - - + Fig.9
140 WT sulA lexA sulA lexA 120
r
e
b 100
m
u
n
80
e
a
l
l
i
p 60
a
P 40
20
0 TnsABC +123456789101112 ++ + + + + + TnsE - - + - - + - - + - - + Fig.10
origin origin DSB A RecBCD
Arm too long Fork stall site for RecBCD terminus far from DSB to degrade New fork encounters the previous fork at stall site
origin origin DSB B RecBCD
Fork stall site RecBCD close to DSB degrades arm terminus