Replication Fork Reversal after Replication–Transcription Collision

Replication fork arrest is a recognized source of genetic instability, and transcription is one of the most prominent causes of replication impediment. We analyze here the requirement for recombination proteins in Escherichia coli when replication–transcription head-on collisions are induced at a specific site by the inversion of a highly expressed ribosomal operon (rrn). RecBC is the only recombination protein required for cell viability under these conditions of increased replication-transcription collisions. In its absence, fork breakage occurs at the site of collision, and the resulting linear DNA is not repaired and is slowly degraded by the RecJ exonuclease. Lethal fork breakage is also observed in cells that lack RecA and RecD, i.e. when both homologous recombination and the potent exonuclease V activity of the RecBCD complex are inactivated, with a slow degradation of the resulting linear DNA by the combined action of the RecBC helicase and the RecJ exonuclease. The sizes of the major linear fragments indicate that DNA degradation is slowed down by the encounter with another rrn operon. The amount of linear DNA decreases nearly two-fold when the Holliday junction resolvase RuvABC is inactivated in recB, as well as in recA recD mutants, indicating that part of the linear DNA is formed by resolution of a Holliday junction. Our results suggest that replication fork reversal occurs after replication–transcription head-on collision, and we propose that it promotes the action of the accessory replicative helicases that dislodge the obstacle

Fork breakage is partially RuvABC-dependent in InvA mutants.

<p>A. Chromosomes of the indicated InvA mutants, grown in minimal medium (MM) or for 1, 2 or 3 hours in LB, were restricted with the <i>Not</i>I enzyme and fragments were separated by PFGE. A. Ethidium bromide stained gel with <i>Not</i>I restricted chromosomes from InvA <i>recB</i> and InvA <i>recB ruvAB</i> mutants. Fragment sizes are indicated on the left. The star indicates the position of migration of the 171 kb DNA fragment formed by fork breakage and DNA degradation. B. Southern blot of the gel shown in A using the <i>rrnC</i> promoter probe, both the intact 208 kb <i>Not</i>I fragment and fragments of smaller sizes hybridize with the probe (the minor hybridization with the 193 kb <i>Not</i>1 restriction fragment may result from co-migration of broken DNA with this fragment). C. Southern blot made with the gel shown in A, using the <i>rrnC</i> terminator probe, only the intact 208 kb <i>Not</i>I fragment hybridizes with the probe. D. Schematic representation of the different DNA fragments. The triangles represent the two <i>rrn</i> operons as indicated, the black circle represents <i>oriC</i> and the bars above the lines show the positions of the probes. E. For each mutant, Southern hybridizations of 3 to 6 gels were quantified, and the percentage of hybridized DNA that remains in wells, that migrates at the 208 kb position and that migrates at the 171 kb position were calculated. For clarity, only the percentages of migrating DNA are shown here (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002622#pgen.1002622.s003" target="_blank">Table S2</a> for complete results); light grey, 208 kb fragment (intact), dark grey, 171 kb fragment (resulting from fork breakage and DNA degradation up to <i>rrnC</i>). Vertical bars indicate standard deviations.</p

Chromosome breakage in InvA <i>recB</i> and InvA <i>recA recD</i> mutants.

<p>Chromosomes of InvA recombination mutants, grown in minimal medium (MM) or for 1, 2 or 3 hours in LB, were cleaved with the I-<i>Sce</i>I enzyme and fragments were separated by PFGE. A. Ethidium bromide stained gel with I-<i>Sce</i>I cut chromosomes from InvA <i>recB</i> and InvA <i>recB recJ</i> mutants. First lane <i>Saccharomyces cerevisiae</i> chromosome ladder, relevant sizes are indicated on the left. B. Southern blot of the gel shown in A using the origin-proximal probe in the 800 kb I-<i>Sce</i>I fragment, both the intact fragment and fragments of smaller sizes hybridize with the probe. C. Southern blot of the gel shown in A using the origin-distal probe in the 800 kb I-<i>Sce</i>I fragment, only the intact I-<i>Sce</i>I fragment hybridizes with the probe. D. Ethidium bromide stained gel with I-<i>Sce</i>I cut chromosomes from the InvA <i>recA recD</i> mutant. E. Southern blot of the gel shown in D using the origin-proximal probe in the 800 kb I-<i>Sce</i>I fragment, both the intact fragment and fragments of smaller sizes hybridize with the probe. F. Schematic representation of the different DNA fragments. The triangles represent the two <i>rrn</i> operons as indicated, the black circle represent <i>oriC</i> and the little bars above the lines represent the position of the origin-proximal (left) and origin distal (right) probes.</p

Inv <i>recB</i> strains are LB–sensitive.

<p>Appropriate dilutions of overnight cultures grown at 37°C in MM (OD 0.8 to 1.5) were plated on MM and LB plates, which were incubated at 37°C. White boxes: colony forming units (cfu)/ml on MM plates after 48 h incubation; grey boxes: cfu/ml on LB plates after 16–24 h incubation. Bars indicate standard deviations. Top: InvBE strains; bottom: InvA strains. <i>ruv</i> stands for <i>ruvAB</i> inactivation. <i>rpo</i>* stands for the <i>rpoC</i>^Δ215–220 mutation, pR stands for pEM001, the plasmid encoding RNaseH. Colonies were small in 48 h on MM for Inv <i>recB ruv</i> and Inv <i>recB ruv recG</i> mutants, but a similar growth delay was observed for non-inverted strains. A small percentage of small colonies was observed after two to four days of incubation on LB with the InvBE <i>recB ruvAB</i> (or <i>recG</i>) and InvBE <i>recB ruvAB recG</i> mutants, however the number of these colonies was highly variable and no colony was ever observed with the InvA <i>recB ruvAB</i> (and/or <i>recG</i>) mutants, indicating that these mutants still require RecBC for growth on rich medium.</p

Chromosome breakage in InvBE <i>recB</i> and InvA <i>recA recD</i> mutants.

<p>Chromosomes of the indicated InvBE mutants, grown in minimal medium (MM) or for 1, 2 or 3 hours in LB, were cleaved with the I-<i>Sce</i>I enzyme and fragments were separated by PFGE. A. Ethidium bromide stained gel with I-<i>Sce</i>I cut chromosomes from InvBE <i>recB</i> and InvBE <i>recB recJ</i> mutants. First lane <i>Saccharomyces cerevisiae</i> chromosome ladder, relevant sizes are indicated on the left. B. Southern blot of the gel shown in A using the origin-proximal probe in the 800 kb I-<i>Sce</i>I fragment, both the intact fragment and fragments of smaller sizes hybridize with the probe. C. Southern blot of a gel made with the InvBE <i>recB</i> I-<i>Sce</i>I cut chromosomes, using the origin-distal probe in the 800 kb I-SceI fragment, only the intact I-<i>Sce</i>I fragment hybridizes with the probe. D. Ethidium bromide stained gel with I-<i>Sce</i>I cut chromosomes from the InvBE <i>recA recD</i> mutant. E. Southern blot of the gel shown in D using the origin-proximal probe in the 800 kb I-<i>Sce</i>I fragment, both the intact fragment and fragments of smaller sizes hybridize with the probe. F. Schematic representation of the different DNA fragments. The triangles represent the three <i>rrn</i> operons as indicated, the black circle represent <i>oriC</i> and the little bars above the lines represent the position of the origin-proximal (left) and origin-distal (right) probes.</p

<i>rrn</i> operons are a barrier to DNA degradation.

<p>Top; schematic representation of the I-<i>Sce</i>I fragment carrying the inverted <i>rrnE</i> operon. Triangles represent <i>rrn</i> operons as indicated, the black circle represents <i>oriC</i>, the black line represents the DNA I-<i>Sce</i>I fragment and the numbered bars under this line show the positions of the different probes. Bottom; chromosomes of InvBE <i>recA recD</i> cells grown in MM or in LB for 2 hours were cleaved with I-<i>Sce</i>I and fragments were separated by PFGE, for each panel: left lane, cells grown in MM, right lane, cells grown in LB for 2 hours. Southern blots were hybridized with the different probes indicated above each panel. From left to right: probe 1 - origin-proximal probe, probe 2 - <i>rrnC</i> promoter probe, probe 3 - <i>rrnC</i> terminator probe, probe 4 - <i>rrnA</i> promoter probe, probe 5 - <i>rrnA</i> terminator probe, probe 6 - origin-distal probe. A schematic representation of the fragments of different length is shown on the right. For each probe all hybridizing fragments are necessarily larger than the distance between the origin-proximal I-<i>Sce</i>I site and the probe, so that the smear stops at the position of the probe.</p

Immunoglobulin gene analysis in chronic lymphocytic leukemia in the era of next generation sequencing

Twenty years after landmark publications, there is a consensus that the somatic hypermutation (SHM) status of the clonotypic immunoglobulin heavy variable (IGHV) gene is an important cornerstone for accurate risk stratification and therapeutic decision-making in patients with chronic lymphocytic leukemia (CLL). The IGHV SHM status has traditionally been determined by conventional Sanger sequencing. However, NGS has heralded a new era in medical diagnostics and immunogenetic analysis is following this trend. There is indeed a growing demand for shifting practice and using NGS for IGHV gene SHM assessment, although it is debatable whether it is always justifiable, at least taking into account financial considerations for laboratories with limited resources. Nevertheless, as this analysis impacts on treatment decisions, standardization of both technical aspects, and data interpretation becomes essential. Also, the need for establishing new recommendations and providing dedicated education and training on NGS-based immunogenetics is greater than ever before. Here we address potential and challenges of NGS-based immunogenetics in CLL. We are convinced that this perspective helps the hematological community to better understand the pros and cons of this new technological development for CLL patient management

Transcription-replication encounters, consequences and genomic instability

To ensure accurate duplication of genetic material, the replication fork must overcome numerous natural obstacles on its way, including transcription complexes engaged along the same template. Here we review the various levels of interdependence between transcription and replication processes and how different types of encounters between RNA-and DNA-polymerase complexes may result in clashes of those machineries on the DNA template and thus increase genomic instability. In addition, we summarize strategies evolved in bacteria and eukaryotes to minimize the consequences of collisions, including R-loop formation and topological stresses. © 2013 Nature America, Inc. All rights reserved