(dT-dC)n and (dG-dA)n tracts arrest single stranded DNA replication in vitro

Abstract

Previous in vivo studies have indicated that (dT-dC)n.(dG-dA)n tracts (referred to here as (TC)n.(GA)n), which are widely dispersed in vertebrate genomes, may serve as pause or arrest signals for DNA replication and amplification. To determine whether these repeat elements act as stop signals for DNA replication in vitro, single stranded DNAs including (TC)n or (GA)n tracts of various lengths, were prepared by cloning such tracts into phage M13 vectors, and were replicated with the Klenow fragment of the E. coli DNA polymerase I, or with the calf thymus DNA polymerase alpha, by extension of an M13 primer. Gel electrophoresis of the reaction products revealed that the replication was specifically arrested around the middle of both (TC)n and (GA)n tracts of n greater than or equal to 16. However, whereas in the (TC)n tracts the arrests were less prominent at pH = 8.0 than at pH = 6.5-7.5, and were completely eliminated at pH = 8.5, the arrests in the (GA)n tracts were stronger at the higher pH values. These results, and previous data, suggest that the arrests were caused by formation of unusual DNA structures, possibly triple helices between partially replicated (TC)n or (GA)n tracts, and unreplicated portions of these sequences.