TY - JOUR
T1 - Precise mapping and characterization of the RNA primers of DNA replication for a yeast hypersuppressive petite by in vitro capping with guanylyltransferase
AU - Graves, Tina
AU - Dante, Michael
AU - Eisenhour, Lynn
AU - Christianson, Thomas W.
N1 - Funding Information:
We thank Ronald Butow (Department of Biochemistery, University of Texas South Medical Center, Dallas, TX) for generously providing the petite HS40. This work was supported by the US Public Health Service, NIH grant GM46009.
PY - 1998/3/1
Y1 - 1998/3/1
N2 - The active origins of DNA replication for yeast (Saccharomyces cerevisiae) mitochondrial DNA share 280 conserved base pairs and have a promoter. Since intact replication intermediates retain their initiating ribonucleotide triphosphate, we used guanylyltransferase to in vitro cap the replication intermediates present in restriction enzyme-cut DNA from an ori-5 hypersuppressive petite. Restriction mapping and RNA sequencing of these labeled intermediates showed that each DNA strand is primed at a single discrete nucleotide, that one primer starts at the promoter and that the other primer starts 34 nt away, outside the conserved region. Deoxyribonuclease digestion of the capped fragments left resistant RNA primers, which enabled identification of zones of transition from RNA to DNA synthesis. Some of the results contradict the prevailing model for priming at the yeast mitochondrial origins.
AB - The active origins of DNA replication for yeast (Saccharomyces cerevisiae) mitochondrial DNA share 280 conserved base pairs and have a promoter. Since intact replication intermediates retain their initiating ribonucleotide triphosphate, we used guanylyltransferase to in vitro cap the replication intermediates present in restriction enzyme-cut DNA from an ori-5 hypersuppressive petite. Restriction mapping and RNA sequencing of these labeled intermediates showed that each DNA strand is primed at a single discrete nucleotide, that one primer starts at the promoter and that the other primer starts 34 nt away, outside the conserved region. Deoxyribonuclease digestion of the capped fragments left resistant RNA primers, which enabled identification of zones of transition from RNA to DNA synthesis. Some of the results contradict the prevailing model for priming at the yeast mitochondrial origins.
UR - http://www.scopus.com/inward/record.url?scp=0032030314&partnerID=8YFLogxK
U2 - 10.1093/nar/26.5.1309
DO - 10.1093/nar/26.5.1309
M3 - Article
C2 - 9469842
AN - SCOPUS:0032030314
VL - 26
SP - 1309
EP - 1316
JO - Nucleic Acids Research
JF - Nucleic Acids Research
SN - 0305-1048
IS - 5
ER -