Regulation of HPV18 Genome Replication, Establishment and Persistence by Sequences in the Viral Upstream Regulatory Region

During persistent human papillomavirus infection, the viral genome replicates as an extrachromosomal plasmid that is efficiently partitioned to daughter cells during cell division. We have previously shown that an element which overlaps the HPV18 transcriptional enhancer promotes stable DNA replication of replicons containing the viral replication origin. Here we perform comprehensive analyses to elucidate the function of this maintenance element. We conclude that no unique element or binding site in this region is absolutely required for persistent replication and partitioning, and instead propose that the overall chromatin architecture of this region is important to promote efficient use of the replication origin. These results have important implications on the genome partitioning mechanism of papillomaviruses. Importance Persistent infection with oncogenic HPVs is responsible for ∼5% human cancers. The viral DNA replicates as an extrachromosomal plasmid and is partitioned to daughter cells in dividing keratinocytes. Using a complementation assay that allows us to separate viral transcription and replication, we provide insight into viral sequences that are required for long term replication and persistence in keratinocytes. Understanding how viral genomes replicate persistently for such long periods of time will guide the development of anti-viral therapies.

Sequences in the Human papillomavirus Type 18 (HPV18) Upstream Regulatory Region regulate Viral Genome Replication, Establishment and Persistence

During persistent human papillomavirus infection, the viral genome replicates as an extrachromosomal plasmid that is efficiently partitioned to daughter cells during cell division. We have previously shown that an element which overlaps the HPV18 transcriptional enhancer promotes stable DNA replication of replicons containing the viral replication origin. Here we perform comprehensive analyses to elucidate the function of this maintenance element. We conclude that no unique element or binding site in this region is absolutely required for persistent replication and partitioning, and instead propose that the overall chromatin architecture of this region is important to promote efficient use of the replication origin. These results have important implications on the genome partitioning mechanism of papillomaviruses.

Laboratory Evolution Experiments Help Identify a Predominant Region of Constitutive Stable DNA Replication Initiation

ABSTRACT The bacterium Escherichia coli can initiate replication in the absence of the replication initiator protein DnaA and/or the canonical origin of replication oriC in a ΔrnhA background. This phenomenon, which can be primed by R-loops, is called constitutive stable DNA replication (cSDR). Whether DNA replication during cSDR initiates in a stochastic manner through the length of the chromosome or at specific sites and how E. coli can find adaptations to loss of fitness caused by cSDR remain inadequately answered. We use laboratory evolution experiments of ΔrnhA-ΔdnaA strains followed by deep sequencing to show that DNA replication preferentially initiates within a broad region located ∼0.4 to 0.7 Mb clockwise of oriC. This region includes many bisulfite-sensitive sites, which have been previously defined as R-loop-forming regions, and includes a site containing sequence motifs that favor R-loop formation. Initiation from this region would result in head-on replication-transcription conflicts at rRNA loci. Inversions of these rRNA loci, which can partly resolve these conflicts, help the bacterium suppress the fitness defects of cSDR. These inversions partially restore the gene expression changes brought about by cSDR. The inversion, however, increases the possibility of conflicts at essential mRNA genes, which would utilize only a minuscule fraction of RNA polymerase molecules, most of which transcribe rRNA genes. Whether subsequent adaptive strategies would attempt to resolve these conflicts remains an open question. IMPORTANCE The bacterium E. coli can replicate its DNA even in the absence of the molecules that are required for canonical replication initiation. This often requires the formation of RNA-DNA hybrid structures and is referred to as constitutive stable DNA replication (cSDR). Where on the chromosome does cSDR initiate? We answer this question using laboratory evolution experiments and genomics and show that selection favors cSDR initiation predominantly at a region ∼0.6 Mb clockwise of oriC. Initiation from this site will result in more head-on collisions of DNA polymerase with RNA polymerase operating on rRNA loci. The bacterium adapts to this problem by inverting a region of the genome including several rRNA loci such that head-on collisions between the two polymerases are minimized. Understanding such evolutionary strategies in the context of cSDR can provide insights into the potential causes of resistance to antibiotics that target initiation of DNA replication.

Laboratory Evolution Experiments Help Identify a Predominant Region of Constitutive Stable DNA Replication Initiation

The bacterium E. coli can initiate replication in the absence of the replication initiator protein DnaA and / or the canonical origin of replication oriC in a ΔrnhA background. This phenomenon, which can be primed by R-loops, is called constitutive stable DNA replication (cSDR). Whether DNA replication during cSDR initiates in a stochastic manner through the length of the chromosome or at specific sites, and how E. coli can find adaptations to loss of fitness caused by cSDR remain inadequately answered. We use laboratory evolution experiments of ΔrnhA-ΔdnaA followed by deep sequencing to show that DNA replication preferentially initiates within a broad region located ∼0.4-0.7 Mb clockwise of oriC. This region includes many bisulfite-sensitive sites, which have been previously defined as R-loop forming regions; and includes a site containing sequence motifs that favour R-loop formation. Initiation from this region would result in head-on replication-transcription conflicts at rRNA loci. Inversions of these rRNA loci, which can partly resolve these conflicts, help the bacterium suppress the fitness defects of cSDR. These inversions partially restore the gene expression changes brought about by cSDR. The inversion however increases the possibility of conflicts at essential mRNA genes, which would utilise only a miniscule fraction of RNA polymerase molecules most of which transcribe rRNA genes. Whether subsequent adaptive strategies would attempt to resolve these conflicts remains an open question.ImportanceThe bacterium E. coli can replicate its DNA even in the absence of the molecules that are required for canonical replication initiation. This often requires the formation of RNA-DNA hybrid structures, and is referred to as constitutive stable DNA replication (cSDR). Where on the chromosome does cSDR initiate? We answer this question using laboratory evolution experiments and genomics, and show that selection favours cSDR initiation predominantly at a region ∼0.6 Mb clockwise of oriC. Initiation from this site will result in more head on collisions of DNA polymerase with RNA polymerase operating on rRNA loci. The bacterium adapts to this problem by inverting a region of the genome including several rRNA loci such that head-on collisions between the two polymerases are minimised. Understanding such evolutionary strategies in the context of cSDR can provide insights into the potential causes of resistance against antibiotics that target initiation of DNA replication.

Stable DNA replication: interplay between DNA replication, homologous recombination, and transcription

Chromosome replication in Escherichia coli is normally initiated at oriC, the origin of chromosome replication. E. coli cells possess at least three additional initiation systems for chromosome replication that are normally repressed but can be activated under certain specific conditions. These are termed the stable DNA replication systems. Inducible stable DNA replication (iSDR), which is activated by SOS induction, is proposed to be initiated from a D-loop, an early intermediate in homologous recombination. Thus, iSDR is a form of recombination-dependent DNA replication (RDR). Analysis of iSDR and RDR has led to the proposal that homologous recombination and double-strand break repair involve extensive semiconservative DNA replication. RDR is proposed to play crucial roles in homologous recombination, double-strand break repair, restoration of collapsed replication forks, and adaptive mutation. Constitutive stable DNA replication (cSDR) is activated in mhA mutants deficient in RNase HI or in recG mutants deficient in RecG helicase. cSDR is proposed to be initiated from an R-loop that can be formed by the invasion of duplex DNA by an RNA transcript, which most probably is catalyzed by RecA protein. The third form of SDR is nSDR, which can be transiently activated in wild-type cells when rapidly growing cells enter the stationary phase. This article describes the characteristics of these alternative DNA replication forms and reviews evidence that has led to the formulation of the proposed models for SDR initiation mechanisms. The possible interplay between DNA replication, homologous recombination, DNA repair, and transcription is explored.