Inferring the DNA replication origin density landscape in human cells from replication timing and fork directionality data

The determinants of the locations and firing times of the multiple replication origins are still elusive in human and other metazoan organisms. Experiments can independently profile mean replication timing (MRT) and replication fork directionality (RFD) genome-wide. In the hypothesis of a constant replication fork speed, MRT and RFD are related to each other by an analytical formula so are a priori equivalent. However, we show here that experimental noises result in MRT and RFD profiles containing information at different spatial frequencies. We further demonstrate that one can compute an origin density landscape that, when inserted in an appropriate simulation framework, jointly predicts experimental MRT and RFD profiles with an unprecedented precision. We also extract an analytical formula linking intrinsic origin efficiency with observed origin efficiency and MRT. We then compare the computed origin density landscape with experimental distributions of potential origins (ORC, MCM) or actual initiation events (Bubble-seq, SNS-seq, OK-seq). The results indicate that MRT and RFD data are highly consistent with each other, that our simple model suffices to capture the replication dynamics during S phase given an appropriate initiation probability landscape, but that the density of potential origins is not the sole determinant of this landscape.

Can a nick promote partial genome re-replication?

Single-stranded DNA breaks, including simple nicks, are amongst the most common forms of DNA damage in cells. They can be readily repaired by ligation; however, if a nick occurs just ahead of an approaching replisome, the outcome is a ‘collapsed’ replication fork in which the nick is converted into a single-ended double-strand DNA break. Attention has largely focused on the processes by which this broken end is used to prime replication restart. We realized that in eukaryotic cells, where replication initiates from multiple replication origins, a second fork converging on the collapsed fork offers additional opportunities for repair, but also generates a substrate that can promote localized re-replication. We have modelled this with purified proteins in vitro and have found that there is, indeed, an additional hazard that eukaryotic replisomes face. We discuss how this problem might be mitigated.

A Low Fidelity Virus Shows Increased Recombination during the Removal of an Alphavirus Reporter Gene

Reporter genes for RNA viruses are well-known to be unstable due to putative RNA recombination events that excise inserted nucleic acids. RNA recombination has been demonstrated to be co-regulated with replication fidelity in alphaviruses, but it is unknown how recombination events at the minority variant level act, which is important for vaccine and trans-gene delivery design. Therefore, we sought to characterize the removal of a reporter gene by a low-fidelity alphavirus mutant over multiple replication cycles. To examine this, GFP was inserted into TC-83, a live-attenuated vaccine for the alphavirus Venezuelan equine encephalitis virus, as well as a low-fidelity variant of TC-83, and passaged until fluorescence was no longer observed. Short-read RNA sequencing using ClickSeq was performed to determine which regions of the viral genome underwent recombination and how this changed over multiple replication cycles. A rapid removal of the GFP gene was observed, where minority variants in the virus population accumulated small deletions that increased in size over the course of passaging. Eventually, these small deletions merged to fully remove the GFP gene. The removal was significantly enhanced during the passaging of low-fidelity TC-83, suggesting that increased levels of recombination are a defining characteristic of this mutant.

Measuring Alphavirus Fidelity Using Non-Infectious Virus Particles

Mutations are incorporated into the genomes of RNA viruses at an optimal frequency and altering this precise frequency has been proposed as a strategy to create live-attenuated vaccines. However, determining the effect of specific mutations that alter fidelity has been difficult because of the rapid selection of the virus population during replication. By deleting residues of the structural polyprotein PE2 cleavage site, E3Δ56-59, in Venezuelan equine encephalitis virus (VEEV) TC-83 vaccine strain, non-infectious virus particles were used to assess the effect of single mutations on mutation frequency without the interference of selection that results from multiple replication cycles. Next-generation sequencing analysis revealed a significantly lower frequency of transversion mutations and overall mutation frequency for the fidelity mutants compared to VEEV TC-83 E3Δ56-59. We demonstrate that deletion of the PE2 cleavage site halts virus infection while making the virus particles available for downstream sequencing. The conservation of the site will allow the evaluation of suspected fidelity mutants across alphaviruses of medical importance.

Isolation and analysis of rereplicated DNA by Rerep-Seq

Changes in gene copy number contribute to genomic instability, the onset and progression of cancer, developmental abnormalities and adaptive potential. The origins of gene amplifications have remained elusive; however, DNA rereplication has been implicated as a source of gene amplifications. The inability to determine which sequences are rereplicated and under what conditions have made it difficult to determine the validity of the proposed models. Here we present Rerep-Seq, a technique that selectively enriches for rereplicated DNA in preparation for analysis by DNA sequencing that can be applied to any species. We validated Rerep-Seq by simulating DNA rereplication in yeast and human cells. Using Rerep-Seq, we demonstrate that rereplication induced in Saccharomyces cerevisiae by deregulated origin licensing is non-random and defined by broad domains that span multiple replication origins and topological boundaries.

Population- and Variant-Based Genome Analyses of Viruses from Vaccine-Derived Rabies Cases Demonstrate Product Specific Clusters and Unique Patterns

Rabies in wildlife has been successfully controlled in parts of Europe and North America using oral rabies vaccination, i.e., the distribution of baits containing live-attenuated virus strains. Occasionally, these vaccines caused vaccine virus-induced rabies cases. To elucidate the mechanisms of genetic selection and the effect of viral populations on these rabies cases, a next generation sequencing approach as well as comprehensive data analyses of the genetic diversity of Street Alabama Dufferin (SAD) and ERA vaccine virus strains and vaccine-induced rabies cases from Canada and several European countries were conducted. As a result, twelve newly generated sets of sequencing data from Canada and Poland were added to a pool of previously investigated samples. While the population-based analysis showed a segregation of viruses of ERA vaccine-induced rabies cases from those of SAD Bern original (SAD Bernorig)-derived rabies cases, the in-depth variant analysis revealed three distinct combinations of selected variants for the ERA vaccine-induced cases, suggesting the presence of multiple replication-competent haplotypes in the investigated ERA-BHK21 vaccine. Our findings demonstrate the potential of a deep sequencing approach in combination with comprehensive analyses on the consensus, population, and variant level.

Identification of Multiple Replication Stages and Origins in the Nucleopolyhedrovirus of Anticarsia gemmatalis

To understand the mechanism of replication used by baculoviruses, it is essential to describe all the factors involved, including virus and host proteins and the sequences where DNA synthesis starts. A lot of work on this topic has been done, but there is still confusion in defining what sequence/s act in such functions, and the mechanism of replication is not very well understood. In this work, we performed an AgMNPV replication kinetics into the susceptible UFL-Ag-286 cells to estimate viral genome synthesis rates. We found that the viral DNA exponentially increases in two different phases that are temporally separated by an interval of 5 h, probably suggesting the occurrence of two different mechanisms of replication. Then, we prepared a plasmid library containing virus fragments (0.5–2 kbp), which were transfected and infected with AgMNPV in UFL-Ag-286 cells. We identified 12 virus fragments which acted as origins of replication (ORI). Those fragments are in close proximity to core genes. This association to the core genome would ensure vertical transmission of ORIs. We also predict the presence of common structures on those fragments that probably recruit the replication machinery, a structure also present in previously reported ORIs in baculoviruses.