RecBCD coordinates repair of two ends at a DNA double-strand break, preventing aberrant chromosome amplification

DNA double-strand break (DSB) repair is critical for cell survival. A diverse range of organisms from bacteria to humans rely on homologous recombination for accurate DSB repair. This requires both coordinate action of the two ends of a DSB and stringent control of the resultant DNA replication to prevent unwarranted DNA amplification and aneuploidy. In Escherichia coli, RecBCD enzyme is responsible for the initial steps of homologous recombination. Previous work has revealed recD mutants to be nuclease defective but recombination proficient. Despite this proficiency, we show here that a recD null mutant is defective for the repair of a two-ended DSB and that this defect is associated with unregulated chromosome amplification and defective chromosome segregation. Our results demonstrate that RecBCD plays an important role in avoiding this amplification by coordinating the two recombining ends in a manner that prevents divergent replication forks progressing away from the DSB site

In vivo single-molecule imaging of RecB reveals efficient repair of DNA damage in Escherichia coli

Efficient DNA repair is essential for maintaining genome integrity and ensuring cell survival. In Escherichia coli, RecBCD plays a crucial role in processing DNA ends, following a DNA double-strand break (DSB), to initiate repair. While RecBCD has been extensively studied in vitro, less is known about how it contributes to rapid and efficient repair in living bacteria. Here, we use single-molecule microscopy to investigate DNA repair in real time in E. coli. We quantify RecB single-molecule mobility and monitor the induction of the DNA damage response (SOS response) in individual cells. We show that RecB binding to DNA ends caused by endogenous processes leads to efficient repair without SOS induction. In contrast, repair is less efficient in the presence of exogenous damage or in a mutant strain with modified RecB activities, leading to high SOS induction. Our findings reveal how subtle alterations in RecB activity profoundly impact the efficiency of DNA repair in E. coli

A perfect palindrome in the Escherichia coli chromosome forms DNA hairpins on both leading- and lagging-strands

DNA palindromes are hotspots for DNA double strand breaks, inverted duplications and intra-chromosomal translocations in a wide spectrum of organisms from bacteria to humans. These reactions are mediated by DNA secondary structures such as hairpins and cruciforms. In order to further investigate the pathways of formation and cleavage of these structures, we have compared the processing of a 460 base pair (bp) perfect palindrome in the Escherichia coli chromosome with the same construct interrupted by a 20 bp spacer to form a 480 bp interrupted palindrome. We show here that the perfect palindrome can form hairpin DNA structures on the templates of the leading- and lagging-strands in a replication-dependent reaction. In the presence of the hairpin endonuclease SbcCD, both copies of the replicated chromosome containing the perfect palindrome are cleaved, resulting in the formation of an unrepairable DNA double-strand break and cell death. This contrasts with the interrupted palindrome, which forms a hairpin on the lagging-strand template that is processed to form breaks, which can be repaired by homologous recombination

DNA synthesis during double-strand break repair in escherichia coli

Efficient and accurate repair of DNA double strand breaks (DSBs) is required to maintain genomic stability in both eukaryotes and prokaryotes. In Escherichia coli, DSBs are repaired by homologous recombination (HR). During this process, DNA synthesis needs to be primed and templated from an intact homologous sequence to restore any information that may have been lost on the broken DNA molecule. Two critical late stages of the pathway are repair DNA synthesis and the processing of Holliday junctions (HJs). However, our knowledge of the detailed mechanisms of these steps is still limited. Our laboratory has developed a system that permits the induction of a site-specific DSB in the bacterial chromosome. This break forms in a replication dependent manner on one of the sister chromosomes, leaving the second sister chromosome intact for repair by HR. Unlike previously available systems, the repairable nature of these breaks has made it possible to physically investigate the different stages of DNA double-strand break repair (DSBR) in a chromosomal context. In this thesis, I have addressed some fundamental questions relating to repair DNA synthesis and processing of HJs by using a combination of mutants defective in specific biochemical reactions and an assay that I have developed to detect repair DNA synthesis, using a polar termination sequence (terB). First, by using terB sites located at different locations around the break point, it was shown that the DnaB-dependent repair forks are established in a coordinated manner, meaning that the collision of the repair forks occurs between two repair DNA synthesis initiation sites. Second, DSBR was shown to require the PriB protein known to transduce the DNA synthesis initiation signal from PriA protein to DnaT. Conversely, the PriC protein (known as an alternative to PriB in some reactions) was not required in this process. PriB was also shown to be required to establish DnaB-dependent repair synthesis using the terB assay. Third, the establishment and termination of repair DNA synthesis by collision of converging repair forks were shown to occur independently of HJ resolution. This conclusion results from the comparison of the viability of single and double mutants, deficient in either the establishment of DNA synthesis, HJ resolution or in both reactions, subjected to DSBs and from the study of the DNA intermediates that accumulated in these mutants as detected by two-dimensional gel electrophoresis. Fourth, the role of RecG protein during DSB repair was investigated. Solexa sequencing analyses showed that recG null mutant cells undergoing DSBs accumulate more DNA around the break point (Mawer and Leach, unpublished data). This phenomenon was further investigated by two different approaches. Using terB sites in different locations around the break point and ChIP-Seq analyses to investigate the distribution of RecA in a recG null mutant demonstrating that the establishment of repair forks depends on the presence of RecG. Further studies using PriA helicase-dead mutant showed that the interplay between RecG and PriA proteins is essential for the establishment of correctly oriented repair forks during DSBR. As a whole, this work provides evidence on the coordinated nature of the establishment and termination of DNA synthesis during DSBR and how this requires a correct interplay between PriA-PriB and RecG. A new adapted model of homologous recombination is presented

Characterizations for the potential operators on Carleson curves in local generalized Morrey spaces

Abstract In this paper, we give a boundedness criterion for the potential operator ℐ α { {\mathcal I} }^{\alpha } in the local generalized Morrey space L M p , φ { t 0 } ( Γ ) L{M}_{p,\varphi }^{\{{t}_{0}\}}(\text{&amp;#x0393;}) and the generalized Morrey space M p , φ ( Γ ) {M}_{p,\varphi }(\text{&amp;#x0393;}) defined on Carleson curves Γ \text{&amp;#x0393;} , respectively. For the operator ℐ α { {\mathcal I} }^{\alpha } , we establish necessary and sufficient conditions for the strong and weak Spanne-type boundedness on L M p , φ { t 0 } ( Γ ) L{M}_{p,\varphi }^{\{{t}_{0}\}}(\text{&amp;#x0393;}) and the strong and weak Adams-type boundedness on M p , φ ( Γ ) {M}_{p,\varphi }(\text{&amp;#x0393;}) .</jats:p

Sharp Inequalities for Driving Point Impedance Functions

In this brief, positive real functions are considered as driving point impedance functions, Z(s), which are utilized in electrical engineering for characteristic representation of circuits. Accordingly, for the real part of Z(s), sharp inequalities are presented with their extremal functions by using zeros and poles of Z(s). For these extremal functions, corresponding generic circuit schematics are given with their spectral analyses. According to the obtained results, it has been shown that various circuits can be obtained by using the extremal functions of the inequalities presented in this brief

Distribution of Holliday junctions and repair forks during Escherichia coli DNA double-strand break repair

Accurate repair of DNA double-strand breaks (DSBs) is crucial for cell survival and genome integrity. In Escherichia coli, DSBs are repaired by homologous recombination (HR), using an undamaged sister chromosome as template. The DNA intermediates of this pathway are expected to be branched molecules that may include 4-way structures termed Holliday junctions (HJs), and 3-way structures such as D-loops and repair forks. Using a tool creating a site-specific, repairable DSB on only one of a pair of replicating sister chromosomes, we have determined how these branched DNA intermediates are distributed across a DNA region that is undergoing DSB repair. In cells, where branch migration and cleavage of HJs are limited by inactivation of the RuvABC complex, HJs and repair forks are principally accumulated within a distance of 12 kb from sites of recombination initiation, known as Chi, on each side of the engineered DSB. These branched DNA structures can even be detected in the region of DNA between the Chi sites flanking the DSB, a DNA segment not expected to be engaged in recombination initiation, and potentially degraded by RecBCD nuclease action. This is observed even in the absence of the branch migration and helicase activities of RuvAB, RadA, RecG, RecQ and PriA. The detection of full-length DNA fragments containing HJs in this central region implies that DSB repair can restore the two intact chromosomes, into which HJs can relocate prior to their resolution. The distribution of recombination intermediates across the 12kb region beyond Chi is altered in xonA, recJ and recQ mutants suggesting that, in the RecBCD pathway of DSB repair, exonuclease I stimulates the formation of repair forks and that RecJQ promotes strand-invasion at a distance from the recombination initiation sites.</jats:p

Distribution of Holliday junctions and repair forks during Escherichia coli DNA double-strand break repair.

Accurate repair of DNA double-strand breaks (DSBs) is crucial for cell survival and genome integrity. In Escherichia coli, DSBs are repaired by homologous recombination (HR), using an undamaged sister chromosome as template. The DNA intermediates of this pathway are expected to be branched molecules that may include 4-way structures termed Holliday junctions (HJs), and 3-way structures such as D-loops and repair forks. Using a tool creating a site-specific, repairable DSB on only one of a pair of replicating sister chromosomes, we have determined how these branched DNA intermediates are distributed across a DNA region that is undergoing DSB repair. In cells, where branch migration and cleavage of HJs are limited by inactivation of the RuvABC complex, HJs and repair forks are principally accumulated within a distance of 12 kb from sites of recombination initiation, known as Chi, on each side of the engineered DSB. These branched DNA structures can even be detected in the region of DNA between the Chi sites flanking the DSB, a DNA segment not expected to be engaged in recombination initiation, and potentially degraded by RecBCD nuclease action. This is observed even in the absence of the branch migration and helicase activities of RuvAB, RadA, RecG, RecQ and PriA. The detection of full-length DNA fragments containing HJs in this central region implies that DSB repair can restore the two intact chromosomes, into which HJs can relocate prior to their resolution. The distribution of recombination intermediates across the 12kb region beyond Chi is altered in xonA, recJ and recQ mutants suggesting that, in the RecBCD pathway of DSB repair, exonuclease I stimulates the formation of repair forks and that RecJQ promotes strand-invasion at a distance from the recombination initiation sites