Mapping vaccinia virus DNA replication origins at nucleotide level by deep sequencing.
Poxviruses reproduce in the host cytoplasm and encode most or all of the enzymes and factors needed for expression and synthesis of their double-stranded DNA genomes. Nevertheless, the mode of poxvirus DNA replication and the nature and location of the replication origins remain unknown. A current but unsubstantiated model posits only leading strand synthesis starting at a nick near one covalently closed end of the genome and continuing around the other end to generate a concatemer that is subsequently resolved into unit genomes. The existence of specific origins has been questioned because any plasmid can replicate in cells infected by vaccinia virus (VACV), the prototype poxvirus. We applied directional deep sequencing of short single-stranded DNA fragments enriched for RNA-primed nascent strands isolated from the cytoplasm of VACV-infected cells to pinpoint replication origins. The origins were identified as the switching points of the fragment directions, which correspond to the transition from continuous to discontinuous DNA synthesis. Origins containing a prominent initiation point mapped to a sequence within the hairpin loop at one end of the VACV genome and to the same sequence within the concatemeric junction of replication intermediates. These findings support a model for poxvirus genome replication that involves leading and lagging strand synthesis and is consistent with the requirements for primase and ligase activities as well as earlier electron microscopic and biochemical studies implicating a replication origin at the end of the VACV genome.
Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892;
Tatiana G
TG
Senkevich
Research Technologies Section, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT 59840;
Daniel
D
Bruno
Research Technologies Section, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT 59840;
Craig
C
Martens
Research Technologies Section, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT 59840;
Stephen F
SF
Porcella
National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD 20894.
Yuri I
YI
Wolf
Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892; bmoss@nih.gov.
Bernard
B
Moss
D004261:DNA Replication; D004279:DNA, Viral / Q000096:biosynthesis*; D059014:High-Throughput Nucleotide Sequencing; D009711:Nucleotides / Q000235:genetics*; D014616:Vaccinia virus / Q000235:genetics*
D004279:DNA, Viral; D009711:Nucleotides
DNA replication; DNA replication fork; DNA replication origin; Okazaki fragments; vaccinia virus