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+) 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.
Ramanathan
Sunita
S
Department of Chemical Sciences and Biological Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Mumbai 400 005, India.
Chary
Kandala V R
KV
Rao
Basuthkar J
BJ
D001483:Base Sequence; D002412:Cations; D002942:Circular Dichroism; D004247:DNA; D004256:DNA Polymerase I / Q000235:genetics* / Q000378:metabolism*; D005092:Exonucleases / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000737:chemistry; D013696:Temperature; D013997:Time Factors
D002412:Cations; D009841:Oligonucleotides; C051802:triplex DNA; D004247:DNA; D004256:DNA Polymerase I; D005092:Exonucleases; D008274:Magnesium