Positive Torsional Strain Causes the Formation of a Four-way Junction at Replication Forks

Lisa Postow,Chris Ullsperger,Rebecca W Keller,Carlos Bustamante,Alexander V Vologodskii,Nicholas R Cozzarelli

doi:10.1074/jbc.m006736200

Lisa Postow, Chris Ullsperger + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m006736200

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Abstract

The advance of a DNA replication fork requires an unwinding of the parental double helix. This in turn creates a positive superhelical stress, a (+)-DeltaLk, that must be relaxed by topoisomerases for replication to proceed. Surprisingly, partially replicated plasmids with a (+)-DeltaLk were not supercoiled nor were the replicated arms interwound in precatenanes. The electrophoretic mobility of these molecules indicated that they have no net writhe. Instead, the (+)-DeltaLk is absorbed by a regression of the replication fork. As the parental DNA strands re-anneal, the resultant displaced daughter strands base pair to each other to form a four-way junction at the replication fork, which is locally identical to a Holliday junction in recombination. We showed by restriction endonuclease digestion that the junction can form at either the terminus or the origin of replication and we visualized the structure with scanning force microscopy. We discuss possible physiological implications of the junction for stalled replication in vivo.

Highlights

Unwinding of the parental strands by helicases during replication allows DNA polymerases access to their template for the synthesis of complementary strands
As the parental DNA strands re-anneal, the resultant displaced daughter strands base pair to each other to form a four-way junction at the replication fork, which is locally identical to a Holliday junction in recombination
We showed by restriction endonuclease digestion that the junction can form at either the terminus or the origin of replication and we visualized the structure with scanning force microscopy

Summary

EXPERIMENTAL PROCEDURES

Plasmid DNAs—Plasmids pREP83 and pREP48 have been described previously [5], and their names indicate the extent of replication allowed by the placement of Ter sites: for example, 83% of pREP83 will replicate. pREP83 was replicated bidirectionally in vitro and contains the E. coli origin of replication, oriC, and Ter sites to block each fork. PREP83 was replicated bidirectionally in vitro and contains the E. coli origin of replication, oriC, and Ter sites to block each fork. The 5.8-kb, pBR322-based pREP48 was used to generate intermediates in vivo, and contains the unidirectional pUC origin of replication followed by one Ter site after 2.8 kb. PTus [5] is a plasmid which expresses the E. coli Tus protein under the control of the arabinose promoter This protein blocks replication forks at Ter sites. The partially replicated plasmid DNA was purified by gel electrophoresis, followed by electroelution, phenol extraction, and ethanol precipitation. Scanning Force Microscopy (SFM)—Partially replicated plasmid DNA (2.5–3 ng) in 5 ␮M ethidium bromide was incubated for 10 min at room temperature. Images were processed with a standard flatten filter using Nanoscope software

RESULTS

DISCUSSION

Number of molecules