Mapping vaccinia virus DNA replication origins at nucleotide level by deep sequencing

Senkevicha Tatiana G.; Bruno Daniel; Martens CraigPorcella Stephen F.Wolf Yuri I.Moss Bernard

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Mapping vaccinia virus DNA replication origins at nucleotide level by deep sequencing

机译：Mapping vaccinia virus DNA replication origins at nucleotide level by deep sequencing

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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.

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《Proceedings of the National Academy of Sciences of the United States of America》 |2015年第35期|10908-10913|共6页
作者
Senkevicha Tatiana G.; Bruno Daniel; Martens CraigPorcella Stephen F.Wolf Yuri I.Moss Bernard;
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作者单位

Natl Lib Med, Natl Ctr Biotechnol Informat, NIH, Bethesda, MD 20894 USA;

NIAID, Lab Viral Dis, NIH, Bethesda, MD 20892 USA;

NIAID, Res Technol Sect, Rocky Mt Labs, NIH, Hamilton, MT 59840 USA;

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收录信息美国《科学引文索引》(SCI);美国《生物学医学文摘》(MEDLINE);美国《化学文摘》(CA);
原文格式 PDF
正文语种英语
中图分类自然科学总论;
关键词
vaccinia virus; DNA replication; DNA replication fork; DNA replication origin; Okazaki fragments;

Mapping vaccinia virus DNA replication origins at nucleotide level by deep sequencing

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