Duplex DNA from sites of helicase-polymerase uncoupling links non-B DNA structure formation to replicative stress

Abstract

Background: Replication impediments can produce helicase-polymerase uncoupling allowing lagging strand synthesis to continue for as much as 6 kb from the site of the impediment. Materials and Methods: We developed a cloning procedure designed to recover fragments from lagging strand near the helicase halt site. Results: A total of 62% of clones from a p53-deficient tumor cell line (PC3) and 33% of the clones from a primary cell line (HPS-19I) were within 5 kb of a G-quadruplex forming sequence. Analyses of a RACK7 gene sequence, that was cloned multiple times from the PC3 line, revealed multiple deletions in region about 1 kb from the cloned region that was present in a non-B conformation. Sequences from the region formed G-quadruplex and i-motif structures under physiological conditions. Conclusion: Defects in components of non-B structure suppression systems (e.g. p53 helicase targeting) promote replication-linked damage selectively targeted to sequences prone to G-quadruplex and i-motif formation.