Klenow exo^-, as opposed to exo⁺, traverses through G–G:C triplex by melting G–G base pairs

Abstract

G–G base-paired hairpin DNA structures on template strands offer potential "road-blocks" to a traversing polymerase. Klenow polymerase (exo⁺) pauses while replicating through G–G base-paired hairpin DNA due to the generation of G–G:C triplex. However, exonuclease-deficient Klenow traverses through de novo generated G–G:C triplexes leading to full-length C:G duplexes. Alleviation of such road-blocks by exo^- Klenow ensues faster at lower Mg²⁺, a kinetic effect consistent with the role of Mg²⁺ in stabilizing G–G:C triplex fold. The ability of exonuclease-deficient polymerase to go past the de novo generated G–G:C triplexes suggests that the "idling" of exo⁺ polymerase at G–G road-block is due to the reiterative polymerase/exonuclease action. The full-length replication product carrying a C_n–G_n duplex at one end is further "expanded" by exo^- Klenow through C-strand "slippage" leading to the generation of C⁺–G:C triplex, which is exemplified by the premature arrest of the same at low pH that further stabilizes the C⁺–G:C triplex.