Mapping replication fork direction by leading strand analysis

Methods : a Companion to Methods in Enzymology
M I Aladjem, G M Wahl

Abstract

Replication fork polarity methods measure the direction of DNA synthesis by taking advantage of the asymmetric nature of DNA replication. One procedure that has been used on a variety of cell lines from different metazoans relies on the isolation of newly replicated DNA strands in the presence of the protein synthesis inhibitor emetine. Since Okazaki fragments are not synthesized under such conditions, DNA strands produced during continuous exposure to emetine consist mainly of leading strands. In the protocol described, leading strands isolated from emetine treated cells are detected with single-stranded probes representing each strand of the DNA duplex in the region of interest. Hybridization of leading strands to one strand of a cloned genomic template identifies the direction of replication fork movement. If initiation of DNA synthesis occurs from a preferred site, leading strands should diverge from the corresponding initiation region. The leading strand method is particularly useful for mapping initiation in chromosomal loci that do not replicate immediately on entry into S phase and in mapping the replication fork patterns in which candidate initiation regions have not been identified. Cautious interpretation of the resu...Continue Reading

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