E. coli SbcCD and RecA Control Chromosomal Rearrangement Induced by an Interrupted Palindrome

Summary Survival and genome stability are critical characteristics of healthy cells. DNA palindromes pose a threat to genome stability and have been shown to participate in a reaction leading to the formation of inverted chromosome duplications centered around themselves. There is considerable interest in the mechanism of this rearrangement given its likely contribution to genome instability in cancer cells. This study shows that formation of large inverted chromosome duplications can be observed in the chromosome of Escherichia coli. They are formed at the site of a 246 bp interrupted DNA palindrome in the absence of the hairpin nuclease SbcCD and the recombination protein RecA. The genetic requirements for this spontaneous rearrangement are consistent with a pathway involving DNA degradation and hairpin formation, as opposed to a cruciform cleavage pathway. Accordingly, the formation of palindrome-dependent hairpin intermediates can be induced by an adjacent DNA double-stand break.

F2/recD-KO-R2, respectively. The 906bp PCR-mediated coupling of these two fragments was cloned between the PstI and SalI sites of plasmid pTOF24, resulting in the kanamycin sensitive pTOFrecD plasmid.
The pTOFsbcB plasmid was used to introduce an in-frame deletion of the sbcB gene.
Two amplified fragments in the sbcB region were ligated, after PCR-mediated coupling, into the pTOF24 plasmid. The 466bp-upstream fragment and the 438bp-downstream fragment were amplified from E. coli MG1655 using primer pairs sbcB-KO-F1/sbcB-KO-R1 and sbcB-KO-F2/sbcB-KO-R2, respectively. The 880bp PCR-mediated coupling of these two fragments was cloned between the PstI and SalI sites of plasmid pTOF24, resulting in the kanamycin sensitive pTOFsbcB plasmid.
The pTOFrecB plasmid was used to introduce an in-frame deletion of the recB gene.
Two amplified fragments in the recB region were ligated, after PCR-mediated coupling, into the pTOF24 plasmid. The 446bp-upstream fragment and the 460bp-downstream fragment were amplified from E. coli MG1655 using primer pairs recB-KO-F1/recB-KO-R1 and recB-KO-F2/recB-KO-R2, respectively. The 882bp PCR-mediated coupling of these two fragments was cloned between the PstI and SalI sites of plasmid pTOF24, resulting in the kanamycin sensitive pTOFrecB plasmid.
The pYaiOIsceI plasmid was used to introduce an I-SceI cleavage site into the yaiO gene. Two amplified fragments in the yaiO region were ligated, after PCR-mediated coupling, into the pTOF24 plasmid. The 436bp-upstream fragment and the 402bp-downstream fragment were amplified from E. coli MG1655 using primer pairs yaiO1/yaiO2 and yaiO3/yaiO4, respectively. Notably, primers yaiO2 and yaiO3 added an I-SceI restriction site between the two fragments. The 820bp PCR-mediated coupling of these two fragments was cloned between the PstI and SalI sites of plasmid pTOF24, resulting in the kanamycin sensitive pYaiOIsceI plasmid.
In order to introduce a mutation using a derivative of the pTOF24 or pLacD1 plasmid, the specific thermosensitive plasmid was introduced into the E. coli chromosome of the original strain with selection for chloramphenicol resistance. Then, an allele replacement by chromosomal integration and excision was carried out as described by Merlin and collaborators (Merlin et al., 2002). Correct insertion of the mutation was verified by PCR using external primers (or primers Ex-test-F and Ex-test-R for a palindrome). If applicable, the insertion of a restriction site was checked by restriction of the PCR product or deletions were verified by UV sensitivity test.
Specific mutations were transferred into the desired receiving strains using the P1 transduction technique with selection for the appropriate antibiotic resistance (Miller J.H., 1992).
When applicable, the presence of the palindrome was verified by PCR using primers Ex-test-F and Ex-test-R. In order to do the P1 transduction into the MG1655 lacZχ-lacI q lacZ::pal246 cynX::Gm r ΔsbcDC mhpR(EcoRI) ΔrecB (DL4116) strain, the pAM-RecBCD+ plasmid was transiently used to make this strain recombination proficient.

Fluorescence microscopy
Images were acquired at a resolution of 0.129 m per pixel using a Zeiss Axiovert 200 fluorescence microscope equipped with a Photometrics cool-SNAP HQ CCD camera. Stacks of optical section images of CFP and YFP fluorescence were collected and deconvolved using the Autovisualize + Autodeblur program (3D adaptative PSF (blind) deconvolution), then analysed and pseudocoloured using the MetaMorph 6-3r2 program (Molecular Devices).

Plasmids, bacterial strains and primers.
Ts indicates that the plasmid has a temperaturesensitive origin of replication. lacZχ-indicates that a chi sequence situated at 2230bp in the lacZ gene was removed. Restriction sites used for cloning are underlined and sequences required for the PCR-mediated coupling are indicated in bold letters.