TY - JOUR
T1 - The effect of template secondary structure of vaccinia DNA polymerase
AU - Challberg, M. D.
AU - Englund, P. T.
N1 - Copyright:
Copyright 2004 Elsevier B.V., All rights reserved.
PY - 1979
Y1 - 1979
N2 - Vaccinia virus DNA polymerase will utilize a substrate consisting of ΦX174 DNA primed with a strand of unique restriction fragment, but the reaction is inefficient. Examination of the reaction products by alkaline agarose gel electrophoresis revealed a few discrete fragments, each corresponding to an extended primer strand. This result implies that specific barriers exist on the ΦX174 template which impede, but do not completely halt, the progress of the enzyme. Only a few per cent of the template molecules were completely copied. Similar findings were reported by Sherman and Gefter using E. coli DNA polymerase II and fd DNA (J. Mol. Biol. (1976) 103, 61-76). Several observations suggest that the barriers are regions of template secondary structure. Some barriers are more effective than others, and they increase in both effectiveness and number as the temperature is decreased. The same barriers are observed with T4 DNA polymerase, but none are detected with E. coli DNA polymerase I. Finally, the major barriers are located in regions of the ΦX174 sequence known to contain hairpin structures of relatively high stability. The exact stopping point at one of the major barriers is within the duplex stem of a hairpin structure. These results show that DNA polymerases are a useful probe of the secondary structure of a single-stranded DNA.
AB - Vaccinia virus DNA polymerase will utilize a substrate consisting of ΦX174 DNA primed with a strand of unique restriction fragment, but the reaction is inefficient. Examination of the reaction products by alkaline agarose gel electrophoresis revealed a few discrete fragments, each corresponding to an extended primer strand. This result implies that specific barriers exist on the ΦX174 template which impede, but do not completely halt, the progress of the enzyme. Only a few per cent of the template molecules were completely copied. Similar findings were reported by Sherman and Gefter using E. coli DNA polymerase II and fd DNA (J. Mol. Biol. (1976) 103, 61-76). Several observations suggest that the barriers are regions of template secondary structure. Some barriers are more effective than others, and they increase in both effectiveness and number as the temperature is decreased. The same barriers are observed with T4 DNA polymerase, but none are detected with E. coli DNA polymerase I. Finally, the major barriers are located in regions of the ΦX174 sequence known to contain hairpin structures of relatively high stability. The exact stopping point at one of the major barriers is within the duplex stem of a hairpin structure. These results show that DNA polymerases are a useful probe of the secondary structure of a single-stranded DNA.
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M3 - Article
C2 - 381293
AN - SCOPUS:0018728272
SN - 0021-9258
VL - 254
SP - 7820
EP - 7826
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 16
ER -