화학공학소재연구정보센터
Biochemical and Biophysical Research Communications, Vol.295, No.3, 730-736, 2002
Klenow exo(-), as opposed to exo(+), traverses through G-G : C triplex by melting G-G base pairs
G-G base-paired hairpin DNA structures on template strands offer potential "road-blocks" to a traversing polymerase. Klenow polymerase (exo(divided by)) 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. (C) 2002 Elsevier Science (USA). All rights reserved.