MAPPING REPLICATION FORK DIRECTION BY LEADING-STRAND ANALYSIS

Replication fork polarity methods measure the direction of DNA synthes is by taking advantage of the asymmetric nature of DNA replication. On e procedure that has been used on a variety of cell lines from differe nt metazoans relies on the isolation of newly replicated DNA strands i n the presence of the protein synthesis inhibitor emetine. Since Okaza ki fragments are not synthesized under such conditions, DNA strands pr oduced during continuous exposure to emetine consist mainly of leading strands. In the protocol described, leading strands isolated from eme tine treated cells are detected with single-stranded probes representi ng each strand of the DNA duplex in the region of interest. Hybridizat ion of leading strands to one strand of a cloned genomic template iden tifies the direction of replication fork movement. If initiation of DN A synthesis occurs from a preferred site, leading strands should diver ge from the corresponding initiation region. The leading strand method is particularly useful for mapping initiation in chromosomal loci tha t do not replicate immediately on entry into S phase and in mapping th e replication fork patterns in which candidate initiation regions have not been identified. Cautious interpretation of the results is needed because the method relies heavily on quantitative hybridization. Lead ing strand data can be difficult to interpret when the genomic targets are very close to initiation regions and when the targets vary in the ir hybridization efficiency or in the efficiency of incorporation of n ucleotide analogs. The experimental details of the method are reviewed , controls to avoid common pitfalls are suggested, protocols to facili tate the accurate interpretation of the results are provided. (C) 1997 Academic Press.