Klenow exo-, as opposed to exo+, traverses through G-G:C triplex by melting G-G base pairs |
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Authors: | Ramanathan Sunita Chary Kandala V R Rao Basuthkar J |
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Institution: | Department of Chemical Sciences and Biological Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Mumbai 400 005, India. |
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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. |
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Keywords: | Secondary structures G-G hairpin Klenow fragment Slippage G-G:C triplex C+-G:G triplex |
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