RecG directs DNA synthesis during double-strand break repair

Homologous recombination provides a mechanism of DNA double-strand break repair (DSBR) that requires an intact, homologous template for DNA synthesis. When DNA synthesis associated with DSBR is convergent, the broken DNA strands are replaced and repair is accurate. However, if divergent DNA synthesis is established, over-replication of flanking DNA may occur with deleterious consequences. The RecG protein of Escherichia coli is a helicase and translocase that can re-model 3-way and 4-way DNA structures such as replication forks and Holliday junctions. However, the primary role of RecG in live cells has remained elusive. Here we show that, in the absence of RecG, attempted DSBR is accompanied by divergent DNA replication at the site of an induced chromosomal DNA double-strand break. Furthermore, DNA double-stand ends are generated in a recG mutant at sites known to block replication forks. These double-strand ends, also trigger DSBR and the divergent DNA replication characteristic of this mutant, which can explain over-replication of the terminus region of the chromosome. The loss of DNA associated with unwinding joint molecules previously observed in the absence of RuvAB and RecG, is suppressed by a helicase deficient PriA mutation (priA300), arguing that the action of RecG ensures that PriA is bound correctly on D-loops to direct DNA replication rather than to unwind joint molecules. This has led us to put forward a revised model of homologous recombination in which the re-modelling of branched intermediates by RecG plays a fundamental role in directing DNA synthesis and thus maintaining genomic stability

Branch Migration Prevents DNA Loss during Double-Strand Break Repair

The repair of DNA double-strand breaks must be accurate to avoid genomic rearrangements that can lead to cell death and disease. This can be accomplished by promoting homologous recombination between correctly aligned sister chromosomes. Here, using a unique system for generating a site-specific DNA double-strand break in one copy of two replicating Escherichia coli sister chromosomes, we analyse the intermediates of sister-sister double-strand break repair. Using two-dimensional agarose gel electrophoresis, we show that when double-strand breaks are formed in the absence of RuvAB, 4-way DNA (Holliday) junctions are accumulated in a RecG-dependent manner, arguing against the long-standing view that the redundancy of RuvAB and RecG is in the resolution of Holliday junctions. Using pulsed-field gel electrophoresis, we explain the redundancy by showing that branch migration catalysed by RuvAB and RecG is required for stabilising the intermediates of repair as, when branch migration cannot take place, repair is aborted and DNA is lost at the break locus. We demonstrate that in the repair of correctly aligned sister chromosomes, an unstable early intermediate is stabilised by branch migration. This reliance on branch migration may have evolved to help promote recombination between correctly aligned sister chromosomes to prevent genomic rearrangements

2D agarose gel electrophoresis 30 Kb upstream of the DSB.

<p>(A) SalI map of the region surrounding the DSB showing the location of the 4.1 kb <i>ykgK</i> fragment analysed by 2D agarose gel electrophoresis. Coordinates for the SalI restriction fragments detected in previous experiments are given in blue. Coordinates for the BspDI restriction fragment detected by 2D agarose gel electrophoresis are given in purple and the <i>ykgK</i> probe is shown as a black rectangle. The location of the palindrome is shown as a green triangle. The 1.5 kb 3x χ arrays are marked by red lines. OP and OD indicate origin-proximal and origin-distal sides of the break, respectively. (B) 2D agarose gel of Δ<i>ruvAB</i> Δ<i>recG</i> mutants containing (DSB⁺), or not (DSB⁻), the palindrome and grown in the presence of 0.2% arabinose for 60 minutes. Strains used were DL4260 (<i>lacZ</i>::<i>pal</i>) and DL4313 (<i>lacZ</i>⁺). (C) Quantification (represented as mean ± SEM where n = 3) of intermediates accumulated in the strain containing the palindrome (DSB⁺), relative to the strain not containing the palindrome (DSB⁻) and the percentage of 4-way DNA junctions and 3-way DNA junctions accumulated in each strain.</p

Intermediates of DSBR by PFG.

<p>(A) Map of the chromosome showing the three SalI fragments around the DSB. The coordinates of the restriction sites are shown in blue. The palindrome is shown as a green triangle and the 1.5 kb 3x χ arrays are shown as red lines. The relative position of probes are represented by small black rectangles. OP and OD indicate origin-proximal and origin-distal sides of the break, respectively. (B–E) PFGs for Rec⁺(DL4184 and DL4201), Δ<i>ruvAB</i> (DL4243 and DL4257), Δ<i>recG</i> (DL4311 and DL4312) and Δ<i>ruvAB</i> Δ<i>recG</i> (DL4260 and DL4313) strains, respectively. Quantifications are represented as mean ± SEM where n = 3. For each probe, Lane 1 contains DNA isolated from a strain not containing the palindrome, grown for 60 minutes in arabinose (pal⁻ SbcCD⁺ T₆₀). Lane 2 contains DNA from a strain containing the palindrome, grown for 60 minutes in glucose (pal⁺ SbcCD⁻ T₆₀). Lane 3 contains DNA from a strain containing the palindrome, prior to the addition of either glucose or arabinose (pal⁺ SbcCD⁻ T₀). Lane 4 contains DNA from a strain containing the palindrome, grown for 60 minutes in arabinose (pal⁺ SbcCD⁺ T₆₀). ‘Branched’ indicates signal from the well, ‘linear’ indicates signal from the gel. Quantifications are represented as mean ± SEM where n = 3. Statistical analysis was carried out using a paired T-test. * represents p<0.05, ** represents p<0.01 and *** represents p<0.005.</p

Making and repairing a site-specific DNA double-strand break in the <i>E. coli</i> chromosome.

<p>(A) SbcCD-mediated cleavage of a 246 bp interrupted palindrome inserted into the chromosomal <i>lacZ</i> gene. During replication, the palindrome becomes transiently single-stranded on the lagging-strand template. This allows it to form a DNA hairpin that is cleaved by SbcCD, generating a two-ended DSB. OP and OD indicate origin-proximal and origin-distal sides of the break, respectively. The palindrome is highlighted by green arrows. (B) RecBCD-mediated HR. The ends of the break are processed by RecBCD to generate 3′ ssDNA overhangs coated in RecA. RecA searches the genome for a homologous DNA sequence and catalyses strand-invasion. This forms a D-loop and HJs. The D-loop is acted upon by the replisome assembly factor, PriA, which initiates DNA synthesis. The HJs can be acted upon by RuvABC, branch-migrated and resolved. This generates two converging replication forks, which, upon convergence, terminate the repair process. (C) Map of the <i>lacZ</i> region of the <i>E. coli</i> chromosome illustrating the position and sequence of two 3x χ arrays that have been inserted 1.5 Kb either side of the palindrome in order to stimulate recombination in close proximity of the DSB. The 8 bp χ recognition sequence, highlighted in red, is repeated three times. OP and OD indicate origin-proximal and origin-distal sides of the break, respectively. Pal represents the position of the palindrome.</p

Detection of branch migration using PFGE.

<p>(A) Map of the chromosome showing the three SalI fragments around the DSB. The coordinates of the restriction sites are shown in blue. The palindrome is shown as a green triangle and the 1.5 kb 3x χ arrays are shown as red lines. The relative position of probes are represented by small black rectangles. OP and OD indicate origin-proximal and origin-distal sides of the break, respectively. (B) Gel of branched DNA retained in the wells of PFGs from DNA isolated from Δ<i>ruvC</i> (DL4913 and DL4914) mutants. Samples were run as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004485#pgen-1004485-g003" target="_blank">Figure 3</a>. (C) Quantifications (represented as mean ± SEM where n = 3) of branched DNA retained in the wells of PFGs from DNA isolated from Δ<i>ruvAB</i> (DL4243 and DL4257) mutants (gel shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004485#pgen-1004485-g003" target="_blank">Figure 3C</a>) and Δ<i>ruvC</i> mutants (gel shown in panel B). Statistical analysis was carried out using a paired T-test. * represents p<0.05, ** represents p<0.01.</p

Viability of strains containing the palindrome, grown in 0.2% arabinose.

<p>(A) Chronic exposure to DSBs. Serial dilutions of strains were spotted on LB-agar plates supplemented with either 0.2% arabinose or 0.5% glucose and incubated overnight at 37°C. (B) Acute exposure to DSBs. Serial dilutions of strains containing the palindrome and grown in 0.2% arabinose for either 0, 30, 60, or 90 minutes were spotted on LB-agar plates supplemented with 0.5% glucose and incubated overnight at 37°C. Strains used; Rec⁺ Pal⁺ (DL2006), Rec⁺ Pal⁻ (DL2573), Δ<i>recA</i> Pal⁺ (DL2075), Δ<i>recA</i> Pal⁻ (DL2605), Δ<i>ruvAB</i> Pal⁺ (DL2801), Δ<i>ruvAB</i> Pal⁻ (DL2800), Δ<i>recG</i> Pal⁺ (DL2511), Δ<i>recG</i> Pal⁻ (DL2610), Δ<i>ruvAB</i> Δ<i>recG</i> Pal⁺ (DL4464), Δ<i>ruvAB</i> Δ<i>recG</i> Pal⁻ (DL4465).</p

Models of DSBR.

<p>(A) Stabilising DSBR intermediates by branch migration. In <i>E. coli</i>, following extensive DNA degradation by RecBCD, a resected 3′ end invades a sister chromosome to establish a D-loop in a reaction catalysed by RecA protein. This is stabilised via branch migration catalysed by RuvAB or RecG to form a Holliday junction that can be resolved to generate a replication fork. Only one end is shown here, but a two-ended reaction can occur as shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004485#pgen-1004485-g001" target="_blank">Figure 1</a>. In the absence of branch migration (in a Δ<i>ruvAB</i> Δ<i>recG</i> mutant) the products of RecA-mediated strand-invasion (3-way D-loops) are unstable and non-proficient for repair. This results in extrusion of the invading end from the unbroken chromosome to re-generate a broken end. This end is processed by RecBCD and a second round of strand-invasion is initiated. The whole process is repeated. Over time the broken chromosome is degraded. (B) Stabilising DSBR intermediates by second-end capture. In the canonical eukaryotic DSBR pathway for the repair of a two-ended DSB, one of two 3′ ssDNA ends invades an intact DNA duplex, at a region of homology, to generate a 3-way DNA junction (D-loop). DNA synthesis is then primed off the 3′ DNA end and this leads to the extension of the D-loop, which eventually uncovers enough homology to allow second-end capture. This generates a stable dHJ intermediate, which is then resolved to generate the recombinant products of repair. Alternatively, the 3′ invading DNA strand is extended allowing second-end capture and then both invading strands are ejected and re-anneal in a reaction know as Synthesis Dependant Strand Annealing (SDSA). (C) Unstable DSBR intermediates for the repair of a one-ended DSB by BIR (by D-loop migration) in eukaryotic cells. The 3′ ssDNA ends invades an intact DNA duplex, at a region of homology, to generate a 3-way DNA junction (D-loop). DNA synthesis is primed off the 3′ end. As synthesis proceeds, the unstable D-loop migrates with the replication fork, resulting in the extrusion of the newly synthesised strand and conservative DNA replication. Template switching may occur. The reaction ends when the D-loop either reaches the end of a chromosomes or converges with an oncoming replication fork.</p

Detection of DNA loss in Δ<i>ruvAB</i> Δ<i>recG</i> and Δ<i>ruvC</i> Δ<i>recG</i> mutants.

<p>(A) Map of the chromosome showing the three SalI fragments surrounding the DSB. The coordinates of the restriction sites are shown in blue. The palindrome is shown as a green triangle and the 1.5 kb 3x χ arrays are shown as red lines. The relative position of the <i>codB</i> probe is represented by a small black rectangle. OP and OD indicate origin-proximal and origin-distal sides of the break, respectively. (B) Gels probed with <i>codB</i> probe and <i>cysN</i> probe. All strains were grown in the presence of 0.2% arabinose for 60 minutes. DSB⁺ strains contain the palindrome while DSB⁻ strains do not. Strains used were; Rec⁺ (DL4184 and DL4201), Δ<i>ruvAB</i> Δ<i>recG</i> (DL4260 and DL4313), Δ<i>ruvC</i> (DL4913 and DL4914), Δ<i>recG</i> (DL4311 and DL4312), Δ<i>ruvC</i> Δ<i>recG</i> (DL4941 and DL4942). All lanes shown for each probe were derived from the same membrane. (C) Quantification (represented as mean ± SEM where n = 3) of linear and branched DNA relative to Rec⁺. Statistical analysis was carried out using an unpaired T-test. ** represents p<0.01.</p

The RNA-binding protein Puf5 contributes to buffering of mRNA upon chromatin-mediated changes in nascent transcription

Gene expression involves regulation of chromatin structure and transcription, as well as processing of the transcribed mRNA. While there are feedback mechanisms, it is not clear whether these include crosstalk between chromatin architecture and mRNA decay. To address this, we performed a genome-wide genetic screen using a Saccharomyces cerevisiae strain harbouring the H3K56A mutation, which is known to perturb chromatin structure and nascent transcription. We identified Puf5 (also known as Mpt5) as essential in an H3K56A background. Depletion of Puf5 in this background leads to downregulation of Puf5 targets. We suggest that Puf5 plays a role in post-transcriptional buffering of mRNAs, and support this by transcriptional shutoff experiments in which Puf5 mRNA targets are degraded slower in H3K56A cells compared to wild-type cells. Finally, we show that post-transcriptional buffering of Puf5 targets is widespread and does not occur only in an H3K56A mutant, but also in an H3K4R background, which leads to a global increase in nascent transcription. Our data suggest that Puf5 determines the fate of its mRNA targets in a context-dependent manner acting as an mRNA surveillance hub balancing deregulated nascent transcription to maintain physiological mRNA levels