Rapid improvements in sequencing and array-based platforms are resulting in a flood of diverse genome-wide data, including data from exome and whole-genome sequencing, epigenetic surveys, expression profiling of coding and noncoding RNAs, single nucleotide polymorphism (SNP) and copy number profiling, and functional assays. Analysis of these large, diverse data sets holds the promise of a more comprehensive understanding of the genome and its relation to human disease. Experienced and knowledgeable human review is an essential component of this process, complementing computational approaches. This calls for efficient and intuitive visualization tools able to scale to very large data sets and to flexibly integrate multiple data types, including clinical data. However, the sheer volume and scope of data pose a significant challenge to the development of such tools.

Integrative Genomics Viewer

Under conditions of nutrient deprivation or stress, or as cells enter stationary phase, Escherichia coli and related bacteria increase the accumulation of RpoS, a specialized sigma factor. RpoS-dependent gene expression leads to general stress resistance of cells. During rapid growth, RpoS translation is inhibited and any RpoS protein that is synthesized is rapidly degraded. The complex transition from exponential growth to stationary phase has been partially dissected by analyzing the induction of RpoS after specific stress treatments. Different stress conditions lead to induction of specific sRNAs that stimulate RpoS translation or to induction of small-protein antiadaptors that stabilize the protein. Recent progress has led to a better, but still far from complete, understanding of how stresses lead to RpoS induction and what RpoS-dependent genes help the cell deal with the stress.

The RpoS-mediated general stress response in Escherichia coli.

Mutation is the source of genetic diversity on which natural selection acts, therefore understanding the rates of mutations is crucial for understanding evolutionary trajectories. In this Opinion article, the authors discuss how emerging experimental mutation-rate data from genome-wide sequencing studies, combined with population-genetic theory, can provide unifying explanations for the diversity in mutation rates between species and across genomic locations.

Genetic drift, selection and the evolution of the mutation rate

DNA is subject to many endogenous and exogenous insults that impair DNA replication and proper chromosome segregation. DNA double-strand breaks (DSBs) are one of the most toxic of these lesions and must be repaired to preserve chromosomal integrity. Eukaryotes are equipped with several different, but related, repair mechanisms involving homologous recombination, including single-strand annealing, gene conversion, and break-induced replication. In this review, we highlight the chief sources of DSBs and crucial requirements for each of these repair processes, as well as the methods to identify and study intermediate steps in DSB repair by homologous recombination.

/pdf/sources-of-dna-double-strand-breaks-and-models-of-2ej8n7gzfl.pdf

Sources of DNA Double-Strand Breaks and Models of Recombinational DNA Repair

Subject Collection DNA Recombination Models of Recombinational DNA RepairSources of DNA Double-Strand Breaks andAnuja Mehta and James E. HaberMeets DNATranscription and Recombination: When RNAAndres Aguilera and Helene GaillardMechanism and RegulationEnd Resection at Double-Strand Breaks:Lorraine S. SymingtonResection in Homologous RecombinationStructural Studies of DNA End Detection andChristian Linke-Winnebeck, et al.Christian Bernd Schiller, Florian Ulrich Seifert,The Dissolution of Double Holliday JunctionsAnna H. Bizard and Ian D. HicksonFor additional articles in this collection, see http://cshperspectives.cshlp.org/cgi/collection/ Copyright © 2014 Cold Spring Harbor Laboratory Press; all rights reservedDownloaded from http://cshperspectives.cshlp.org/

Sources of DNA Double-Strand Breaks and Models of Recombinational

An important step towards understanding the mechanistic basis of the central dogma is the quantitative characterization of the dynamics of nucleic-acid-bound molecular motors in the context of the living cell. To capture these dynamics, we develop lag-time analysis, a method for measuring in vivo dynamics. Using this approach, we provide quantitative locus-specific measurements of fork velocity, in units of kilobases per second, as well as replisome pause durations, some with the precision of seconds. The measured fork velocity is observed to be both locus and time dependent, even in wild-type cells. In this work, we quantitatively characterize known phenomena, detect brief, locus-specific pauses at ribosomal DNA loci in wild-type cells, and observe temporal fork velocity oscillations in three highly-divergent bacterial species. 

/pdf/the-in-vivo-measurement-of-replication-fork-velocity-and-1hf0dmhs.pdf

The in vivo measurement of replication fork velocity and pausing by lag-time analysis

An important step towards understanding the replication process in the context of the cell is the quantitative characterization of replication dynamics, including the rate of replication fork progression (i.e. fork velocity) with genomic and temporal specificity in vivo. In this paper, we develop a novel method, lag-time analysis, for measuring replisome dynamics using next-generation sequencing. We provide the first quantitative locus-specific measurements of fork velocity. The measured velocity is observed to be both locus and time dependent, even in wild-type cells. To benchmark the approach, we analyze replication dynamics in three different species and a collection of mutants which facilitate the quantitative characterization of replication-conflict-induced fork slowdowns as well as a host of other replication dynamics phenomena, including the observation of temporal fork velocity oscillation. With increases in sequencing depth and improvements in sample preparation, the approach has the potential to provide new insights at higher genomic resolution and in a wide-range of biological systems.

https://www.biorxiv.org/content/biorxiv/early/2022/08/14/2022.08.13.503874.full.pdf

The high-resolution in vivo measurement of replication fork velocity and pausing

Summary

Proper coordination of DNA replication with cell growth and division is critical for production of viable progeny. In bacteria, coordination of DNA replication with cell growth is generally achieved by controlling activity of the replication initiator DnaA and its access to the chromosomal origin of replication, oriC. Here we describe a previously unknown mechanism for regulation of DnaA. YabA, a negative regulator of replication initiation in Bacillus subtilis, interacts with DnaA and DnaN, the sliding (processivity) clamp of DNA polymerase. We found that in vivo, YabA associated with the oriC region in a DnaA-dependent manner and limited the amount of DnaA at oriC. In vitro, purified YabA altered binding of DnaA to DNA by inhibiting cooperativity. Although previously undescribed, proteins that directly inhibit cooperativity may be a common mechanism for regulating replication initiation. Conditions that cause release of DnaN from the replisome, or overproduction of DnaN, caused decreased association of YabA and increased association of DnaA with oriC. This effect of DnaN, either directly or indirectly, is likely responsible, in part, for enabling initiation of a new round of replication following completion of a previous round.

Control of the replication initiator DnaA by an anti-cooperativity factor

Here we show that the core NER factors UvrABC promote spontaneous mutagenesis, across highly divergent bacterial species. We also show that a replicative polymerase, and two Y-family polymerases are responsible for the observed NER-dependent increase in mutagenesis. Critically, our genetic analyses show that all three polymerases function in the same pathway as the NER proteins and Mfd. These findings strongly suggest that bacterial NER is a pro-mutagenic repair pathway that is universally coupled to transcription, and that this is due to the usage of error prone polymerases. Last, we present data that the underlying cause of this process is endogenous and that in general, oxidative damage drives NER mutagenesis and rapid evolution

https://www.biorxiv.org/content/biorxiv/early/2022/06/28/2022.06.28.497968.full.pdf

Nucleotide excision repair is universally mutagenic and 5 transcription-associated 6

Pathogenic bacteria and their eukaryotic hosts are in a constant arms race. Hosts have numerous defense mechanisms at their disposal that not only challenge the bacterial invaders, but have the potential to disrupt molecular transactions along the bacterial chromosome. However, it is unclear how the host impacts association of proteins with the bacterial chromosome at the molecular level during infection. This is partially due to the lack of a method that could detect these events in pathogens while they are within host cells. We developed and optimized a system capable of mapping and measuring levels of bacterial proteins associated with the chromosome while they are actively infecting the host (referred to as PIC-seq). Here, we focused on the dynamics of RNA polymerase (RNAP) movement and association with the chromosome in the pathogenic bacterium Salmonella enterica as a model system during infection. Using PIC-seq, we found that RNAP association patterns with the chromosome change during infection genome-wide, including at regions that encode for key virulence genes. Importantly, we found that infection of a host significantly increases RNAP backtracking on the bacterial chromosome. RNAP backtracking is the most common form of disruption to RNAP progress on the chromosome. Interestingly, we found that the resolution of backtracked RNAPs via the anti-backtracking factors GreA and GreB is critical for pathogenesis, revealing a new class of virulence genes. Altogether, our results strongly suggest that infection of a host significantly impacts transcription by disrupting RNAP movement on the chromosome within the bacterial pathogen. The increased backtracking events have important implications not only for efficient transcription, but also for mutation rates as stalled RNAPs increase the levels of mutagenesis.

/pdf/pathogenic-bacteria-experience-pervasive-rna-polymerase-1qex7b3d.pdf

Houra Merrikh

Papers

The in vivo measurement of replication fork velocity and pausing by lag-time analysis

The high-resolution in vivo measurement of replication fork velocity and pausing

Control of the replication initiator DnaA by an anti-cooperativity factor

Nucleotide excision repair is universally mutagenic and 5 transcription-associated 6

Pathogenic bacteria experience pervasive RNA polymerase backtracking during infection