How living cells deal with head-on collisions of the replication and transcription complexes has been debated for a long time. Even in the widely studied model bacteria Escherichia coli, the enzymes that take care of such collisions are still unknown. We report here that in vivo, the DinG, Rep and UvrD helicases are essential for efficient replication across highly transcribed regions. We show that when rRNA operons (rrn) are inverted to face replication, the viability of the dinG mutant is affected and over-expression of RNase H rescues the growth defect, showing that DinG acts in vivo to remove R-loops. In addition, DinG, Rep and UvrD exert a common function, which requires the presence of two of these three helicases. After replication blockage by an inverted rrn, Rep in conjunction with DinG or UvrD removes RNA polymerase, a task that is fulfilled in its absence by the SOS-induced DinG and UvrD helicases. Finally, Rep and UvrD also act at inverted sequences other than rrn, and promote replication through highly transcribed regions in wild-type E. coli.

We have sequenced the genome of the intracellular symbiont Buchnera aphidicola from the aphid Baizongia pistacea. This strain diverged 80 -150 million years ago from the common ancestor of two previously sequenced Buchnera strains. Here, a field-collected, nonclonal sample of insects was used as source material for laboratory procedures. As a consequence, the genome assembly unveiled intrapopulational variation, consisting of Ϸ1,200 polymorphic sites. Comparison of the 618-kb (kbp) genome with the two other Buchnera genomes revealed a nearly perfect gene-order conservation, indicating that the onset of genomic stasis coincided closely with establishment of the symbiosis with aphids, Ϸ200 million years ago. Extensive genome reduction also predates the synchronous diversification of Buchnera and its host; but, at a slower rate, gene loss continues among the extant lineages. A computational study of protein folding predicts that proteins in Buchnera, as well as proteins of other intracellular bacteria, are generally characterized by smaller folding efficiency compared with proteins of free living bacteria. These and other degenerative genomic features are discussed in light of compensatory processes and theoretical predictions on the long-term evolutionary fate of symbionts like Buchnera.

We assembled genome-wide data from 271 ancient Iberians, of whom 176 are from the largely unsampled period after 2000 BCE, thereby providing a high-resolution time transect of the Iberian Peninsula. We document high genetic substructure between northwestern and southeastern hunter-gatherers before the spread of farming. We reveal sporadic contacts between Iberia and North Africa by~2500 BCE and, by~2000 BCE, the replacement of 40% of Iberia's ancestry and nearly 100% of its Y-chromosomes by people with Steppe ancestry. We show that, in the Iron Age, Steppe ancestry had spread not only into Indo-European-speaking regions but also into non-Indo-European-speaking ones, and we reveal that present-day Basques are best described as a typical Iron Age population without the admixture events that later affected the rest of Iberia. Additionally, we document how, beginning at least in the Roman period, the ancestry of the peninsula was transformed by gene flow from North Africa and the eastern Mediterranean.

DNA synthesis is an accurate and very processive phenomenon; nevertheless, replication fork progression on chromosomes can be impeded by DNA lesions, DNA secondary structures, or DNA-bound proteins. Elements interfering with the progression of replication forks have been reported to induce rearrangements and͞or render homologous recombination essential for viability, in all organisms from bacteria to human. Arrested replication forks may be the target of nucleases, thereby providing a substrate for doublestrand break repair enzyme. For example in bacteria, direct fork breakage was proposed to occur at replication forks blocked by a bona fide replication terminator sequence, a specific site that arrests bacterial chromosome replication. Alternatively, an arrested replication fork may be transformed into a recombination substrate by reversal of the forked structures. In reversed forks, the last duplicated portions of the template strands reanneal, allowing the newly synthesized strands to pair. In bacteria, this reaction was proposed to occur in replication mutants, in which fork arrest is caused by a defect in a replication protein, and in UV irradiated cells. Recent studies suggest that it may also occur in eukaryote organisms. We will review here observations that link replication hindrance with DNA rearrangements and the possible underlying molecular processes. Large genome rearrangements, such as duplications, deletions, translocations and insertions, are mainly catalyzed by three classes of molecular processes that differ by length of homology of the joined sequences. Recombination events formed by joining of homologous sequences are mediated by specific enzymes. The key enzyme, RecA in prokaryotes and RecAhomologues in eukaryotes, catalyzes the strand exchange reaction and is highly conserved from bacteria to human (1, 2). A second type of recombination, called illegitimate, is characterized by the joining sequences whose length is below the minimal length required for recognition of homology by RecA (Minimal Efficient Processing Segment, or MEPS). In prokaryotes, illegitimate recombination can result from simple ligation of unrelated sequences (reviewed in ref.3). In eukaryotes, this process is promoted by a battery of specialized enzymes and is called nonhomologous end-joining (NHEJ; reviewed in refs. 4 and 5). In addition, recombination between tandemly repeated sequences forms a distinct class of events that may be catalyzed by several specific pathways (reviewed in ref. 6). All classes of DNA rearrangements are important in human health, as they may cause cancers (reviewed in ref. 7) or hereditary disorders (reviewed in refs. 8 and 9) and are important in evolution (ref. 10; reviewed in ref. 11). All classes of rearrangements can result from the formation and repair of DNA double-strand breaks (DSBs) and have been shown to occur at an increased frequency in DNA regions difficult to replicate or when DNA replication is affected by a mutation. The correlation between replication hindrance and rearrangem...

Genome rearrangements can take place by a process known as replication slippage or copy-choice recombination. The slippage occurs between repeated sequences in both prokaryotes and eukaryotes, and is invoked to explain microsatellite instability, which is related to several human diseases. We analysed the molecular mechanism of slippage between short direct repeats, using in vitro replication of a single-stranded DNA template that mimics the lagging strand synthesis. We show that slippage involves DNA polymerase pausing, which must take place within the direct repeat, and that the pausing polymerase dissociates from the DNA. We also present evidence that, upon polymerase dissociation, only the terminal portion of the newly synthesized strand separates from the template and anneals to another direct repeat. Resumption of DNA replication then completes the slippage process.

Owing to a typesetting error, Figure 2 of the above article was published incorrectly.The correct figure is reproduced here.The publisher apologizes for this error and any inconvenience it may have caused. Figure 2 The helicases Rep, UvrD and DinG are required for colony formation in Inv mutants. Appropriate dilutions of overnight cultures at 371C in MM (OD 1.0-1.5) were plated on MM and LB plates, which were incubated at 371C. Unmarked positions on the left of (A) (InvA), (B) (InvBE) and (C) (InvABE) are data points for Inv mutants that express all helicases. White boxes: colony forming units (cfu)/ml on MM plates after 48 h incubation; dark grey boxes: cfu/ml on LB plates after 16-24 h incubation; light grey boxes: cfu/ml on LB plates after 48 h of incubation. The hatched box indicates cfu/ml on MM after 3 days incubation. The results are also presented in Supplementary Table S2.

Replication slippage is a particular type of error caused by DNA polymerases believed to occur both in bacterial and eukaryotic cells. Previous studies have shown that deletion events can occur in Escherichia coli by replication slippage between short duplications and that the main E. coli polymerase, DNA polymerase III holoenzyme is prone to such slippage. In this work, we present evidence that the two other DNA polymerases of E. coli, DNA polymerase I and DNA polymerase II, as well as polymerases of two phages, T4 (T4 pol) and T7 (T7 pol), undergo slippage in vitro, whereas DNA polymerase from another phage, ⌽29, does not. Furthermore, we have measured the strand displacement activity of the different polymerases tested for slippage in the absence and in the presence of the E. coli single-stranded DNA-binding protein (SSB), and we show that: (i) polymerases having a strong strand displacement activity cannot slip (DNA polymerase from ⌽29); (ii) polymerases devoid of any strand displacement activity slip very efficiently (DNA polymerase II and T4 pol); and (iii) stimulation of the strand displacement activity by E. coli SSB (DNA polymerase I and T7 pol), by phagic SSB (T4 pol), or by a mutation that affects the 3 3 5 exonuclease domain (DNA polymerase II exo ؊ and T7 pol exo ؊ ) is correlated with the inhibition of slippage. We propose that these observations can be interpreted in terms of a model, for which we have shown that high strand displacement activity of a polymerase diminishes its propensity to slip.Misalignment of two DNA strands during replication can lead to DNA rearrangements such as deletions or duplications of varying lengths ranging from several nucleotides to entire genes. This process, designated replication slippage (as well as copy-choice recombination), has been suspected for a long time to occur both in prokaryotes and eukaryotes between repeated DNA sequences. The process is thought to encompass the following steps: (i) copying of the first duplication by the replication machinery, (ii) replication pausing and dissociation of the polymerase from the newly synthesized end, (iii) unpairing of the newly synthesized strand and its pairing with the second duplication, and (iv) resumption of the DNA synthesis. A heteroduplex is thus formed, containing one parental and one recombinant strand, which are separated by a second round of replication.Replication slippage has been widely proposed as a probable mechanism of genome rearrangements, such as deletions between short duplications in bacteria (1-3), yeast (4), and mammalian mitochondria (5) or deletions between long tandem repeats in Escherichia coli (6 -8), as well as microsatellite instability (for reviews see Refs. 9 -12). Direct evidence for the slippage has been obtained in vivo, in E. coli (13), and in vitro (14). In the latter study, it was shown that E. coli DNA polymerase III holoenzyme (pol III HE), 1 the enzyme that replicates the cell chromosome (for review see Ref. 15), was able to slip, which is of particular significance in vie...

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