PubMed Health⌕ Search
Home/Archive/PubMed

PubMed · 7778304

Minimum origin requirements for linear duplex AAV DNA replication in vitro.

P Ward·K I Berns
View original source ↗Export metadata ↓

Abstract

We have investigated the minimal requirements for a functional origin of DNA replication in an in vitro assay which requires the adeno-associated virus (AAV) Rep 68/78 protein. When a linear duplex template was used, initiation of one round of DNA replication was achieved when nucleotides 1-56 of the AAV inverted terminal repeat (in the flop orientation) were present at the terminus of the template. A terminal resolution site was not required. Deletion of an additional 11 nucleotides from the terminal repeat blocked the reaction.

Explore related subjects

DNA ReplicationDependovirusGenome, ViralModels, GeneticRepetitive Sequences, Nucleic AcidReplication OriginRestriction MappingSequence DeletionTemplates, GeneticComparative StudyJournal ArticleResearch Support, U.S. Gov't, P.H.S.

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

P Ward, K I Berns. 1995-06-01. Minimum origin requirements for linear duplex AAV DNA replication in vitro.. https://doi.org/10.1006/viro.1995.1306

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related citations

PubMed2026

CIZ1 regulates G1 length and the CDK threshold for initiation of DNA replication to prevent DNA replication stress.

Eukaryotic cell division is regulated by CDK activity that must reach critical CDK threshold levels to progress through cell cycle stages. In low-mitogen, low-CDK environments, cells exit the cell cycle into a non-proliferative quiescent state, G0, that plays essential roles in stem cell maintenance and cellular homeostasis. CIZ1 regulates cell cycle and epigenetic programmes, and CIZ1 ablation promotes genomic instability after release from quiescence. Here, we show that CIZ1 contributes to mechanisms that temporally regulate cell cycle transitions in post-quiescent cells. CIZ1-/- (CIZ1 KO) fibroblasts re-entering the cell cycle from quiescence have reduced G1 phase and cell cycle length, mediated by increased intracellular CDK activity and early restriction point bypass via G1/S cyclin overexpression. In addition, CIZ1-/- cells are deficient in cyclin A chromatin binding and require increased CDK activity to initiate DNA replication, leading to DNA replication stress. Importantly, ectopic expression of CIZ1 or addition of recombinant CIZ1 reinstates the CDK threshold for initiation of DNA replication, reversing DNA replication stress and increasing replication fork rates. These data suggest that in post-quiescent cells, CIZ1 determines the threshold CDK activity required for the G1/S transition to prevent DNA replication stress.

DNA Replication↗
PubMed2026

A dominant mutation in tomato DNA POLYMERASE DELTA 1 causes geminivirus DNA replication catastrophe.

Geminiviruses pose a severe threat to grain and vegetable crops worldwide, often resulting in significant economic losses. In cultivated tomato (Solanum lycopersicum), Ty resistance alleles have been introduced from wild tomato relatives, providing partial to strong resistance to geminivirus infections. The Ty-6 resistance locus from Solanum chilense was previously mapped to chromosome 10. It was recently shown to contain a mutant allele of the DNA POLYMERASE DELTA 1 (POLD1) gene that provides resistance to Tomato yellow leaf curl virus (TYLCV) infections. However, the resistance mechanism remained unknown. Here, we report another POLD1 allele at the Ty-6 locus of S. chilense with an E622D mutation in the catalytic site of the POLD1 protein. POLD1E622D is maintained as a heterozygous dominant allele in S. chilense and the AVTO2225 breeding line. It provides full resistance to the severe TYLCV Thailand (TYLCTHV) strain. The E622D amino acid change does not alter the predicted structure of POLD1. Replication of the TYLCTHV genome in plants carrying the POLD1E622D allele is severely compromised by a high frequency of mutations that accumulate in viral DNA, which results in nonfunctional proteins that are essential for continuous viral replication. Ectopically expressing the POLD1E622D allele cDNA alone causes mutations in TYLCTHV genes in inoculated leaves. S. chilense and AVTO2225 plants carrying the POLD1E622D allele mount a hypersensitive response after TYLCTHV infection, indicating that the defective virus genome cannot suppress the plant defense. The dominant POLD1E622D allele is therefore an effective resistance gene that geminiviruses cannot overcome.

DNA Replication↗
PubMed2026

Gabija restricts phage circularization and DNA replication.

Anti-bacteriophage systems such as restriction-modification and CRISPR-Cas have DNA substrate specificity mechanisms that enable the identification of invaders. How Gabija, a highly prevalent nuclease-helicase antiphage system, limits phage replication while executing self- vs. non-self-discrimination remains unknown. Here, we show that phage-encoded DNA end-binding proteins that antagonize host RecBCD sensitize phages to Gabija. When targeting a temperate lambda-like phage in Pseudomonas aeruginosa, Gabija prevents phage genome circularization and subsequent replication. DNA end-binding complexes, including a phage exonuclease and a single-stranded DNA (ssDNA)-annealing protein or GamMu dimers that prevent loading of the host repair complex RecBCD, are necessary and sufficient to license phage and plasmid sensitivity to Gabija. Mutant escape phages lacking these DNA end-binding proteins become protected from Gabija by RecBCD translocation activities. RecBCD activity on the bacterial genome, presumably whenever it is linearized, also prevents Gabija from targeting self-DNA. Therefore, we propose that Gabija antagonizes the circularization and replication of linear DNA devoid of RecBCD as a mechanism to identify and antagonize foreign invaders.

DNA Replication↗