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Type III restriction enzymes cleave DNA by long-range interaction between sites in both head-to-head and tail-to-tail inverted repeat.

ABSTRACT: Cleavage of viral DNA by the bacterial Type III Restriction-Modification enzymes requires the ATP-dependent long-range communication between a distant pair of DNA recognition sequences. The classical view is that Type III endonuclease activity is only activated by a pair of asymmetric sites in a specific head-to-head inverted repeat. Based on this assumption and due to the presence of helicase domains in Type III enzymes, various motor-driven DNA translocation models for communication have been suggested. Using both single-molecule and ensemble assays we demonstrate that Type III enzymes can also cleave DNA with sites in tail-to-tail repeat with high efficiency. The ability to distinguish both inverted repeat substrates from direct repeat substrates in a manner independent of DNA topology or accessory proteins can only be reconciled with an alternative sliding mode of communication.

SUBMITTER: van Aelst K

PROVIDER: S-EPMC2889075 | biostudies-literature | 2010 May

REPOSITORIES: biostudies-literature

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Type III restriction enzymes cleave DNA by long-range interaction between sites in both head-to-head and tail-to-tail inverted repeat.

van Aelst Kara K Tóth Júlia J Ramanathan Subramanian P SP Schwarz Friedrich W FW Seidel Ralf R Szczelkun Mark D MD

Proceedings of the National Academy of Sciences of the United States of America 20100430 20

Cleavage of viral DNA by the bacterial Type III Restriction-Modification enzymes requires the ATP-dependent long-range communication between a distant pair of DNA recognition sequences. The classical view is that Type III endonuclease activity is only activated by a pair of asymmetric sites in a specific head-to-head inverted repeat. Based on this assumption and due to the presence of helicase domains in Type III enzymes, various motor-driven DNA translocation models for communication have been ...[more]

PMID: 20435912

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Project description:DNA replication errors are a major driver of evolutionâ??from single nucleotide polymorphisms to large-scale copy number variations (CNVs). Here we test a specific replication-based model to explain the generation of interstitial, inverted triplications. While no genetic information is lost, the novel inversion junctions and increased copy number of the included sequences create the potential for adaptive phenotypes. The modelâ??Origin-Dependent Inverted-Repeat Amplification (ODIRA)â??proposes that a replication error at pre-existing short, interrupted, inverted repeats in genomic sequences generates an extrachromosomal, inverted dimeric, autonomously replicating intermediate; subsequent genomic integration of the dimer yields this class of CNV without loss of distal chromosomal sequences. We used a combination of in vitro and in vivo approaches to test the feasibility of the proposed replication error and its downstream consequences on chromosome structure in the yeast Saccharomyces cerevisiae. We show that the proposed replication errorâ??the ligation of leading and lagging nascent strands to create a "closed" forkâ??can occur in vitro at short, interrupted inverted repeats. The removal of molecules with closed forks results in a hairpin-capped linear duplex that we show replicates in vivo to create an inverted, dimeric plasmid that subsequently integrates into the genome by homologous recombination, creating an inverted triplication. While other models have been proposed to explain inverted triplications and their derivatives, our model can also explain the generation of human, de novo, inverted amplicons that have a 2:1 mixture of sequences from both homologues of a single parentâ??a feature readily explained by a plasmid intermediate that arises from one homologue and integrates into the other homolog prior to meiosis. Our tests of key features of ODIRA lend support to this mechanism and suggest further avenues of enquiry to unravel the origins of interstitial, inverted CNVs pivotal in human health and evolution These are all CGH arrays comparing DNA copy number between evolved yeast strains and a euploid wt strain.

Project description:Marek's disease virus (MDV) is an oncogenic alphaherpesvirus of chickens. The MDV genome consists of two unique regions that are both flanked by inverted repeat regions. These repeats harbor several genes involved in virus replication and pathogenesis, but it remains unclear why MDV and other herpesviruses harbor these large sequence duplications. In this study, we set to determine if both copies of these repeat regions are required for MDV replication and pathogenesis. Our results demonstrate that MDV mutants lacking the entire internal repeat region (ΔIRLS) efficiently replicate and spread from cell-to-cell in vitro However, ΔIRLS replication was severely impaired in infected chickens and the virus caused significantly less frequent disease and tumors compared to the controls. In addition, we also generated recombinant viruses that harbor a deletion of most of the internal repeat region, leaving only short terminal sequences behind (ΔIRLS-HR). These remaining homologous sequences facilitated rapid restoration of the deleted repeat region, resulting in a virus that caused disease and tumors comparable to the wild type. Therefore, ΔIRLS-HR represents an excellent platform for rapid genetic manipulation of the virus genome in the repeat regions. Taken together, our study demonstrates that MDV requires both copies of the repeats for efficient replication and pathogenesis in its natural host.IMPORTANCE Marek's disease virus (MDV) is a highly oncogenic alphaherpesvirus that infects chickens and causes losses in the poultry industry of up to $2 billion per year. The virus is also widely used as a model to study alphaherpesvirus pathogenesis and virus-induced tumor development in a natural host. MDV and most other herpesviruses harbor direct or inverted repeats regions in their genome. However, the role of these sequence duplications in MDV remains elusive and has never been investigated in a natural virus-host model for any herpesvirus. Here, we demonstrate that both copies of the repeats are needed for efficient MDV replication and pathogenesis in vivo, while replication was not affected in cell culture. With this, we further dissect herpesvirus genome biology and the role of repeat regions in Marek's disease virus replication and pathogenesis.

Dataset Information

Type III restriction enzymes cleave DNA by long-range interaction between sites in both head-to-head and tail-to-tail inverted repeat.

Publications

Type III restriction enzymes cleave DNA by long-range interaction between sites in both head-to-head and tail-to-tail inverted repeat.

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