Synthesis of the mammalian telomere lagging strand in vitro.
Using a synthetic telomere DNA template and whole cell extracts, we have identified proteins capable of synthesizing the telomere complementary strand. Synthesis of the complementary strand required a DNA template consisting of 10 repeats of the human telomeric sequence d(TTAGGG) and deoxy- and ribonucleosidetriphosphates and was inhibited by neutralizing antibodies to DNA polymerase alpha. No evidence for RNA-independent synthesis of the lagging strand was observed, suggesting that a stable DNA secondary structure capable of priming the lagging strand is unlikely. Purified DNA polymerase alpha/primase was capable of catalyzing synthesis of the lagging strand with the same requirements as those observed in crude cell extracts. A ladder of products was observed with an interval of six bases, suggesting a unique RNA priming site and site-specific pausing or dissociation of polymerase alpha on the d(TTAGGG)10 template. Removal of the RNA primers was observed upon the addition of purified RNase HI. By varying the input rNTP, the RNA priming site was determined to be opposite the 3' thymidine nucleotide generating a five-base RNA primer with the sequence 5'-AACCC. The addition of UTP did not increase the efficiency of priming and extension, suggesting that the five-base RNA primer is sufficient for extension with dNTPs by DNA polymerase alpha. This represents the first experimental evidence for RNA priming and DNA extension as the mechanism of mammalian telomeric lagging strand replication.
Reveal
P M
PM
Department of Biochemistry and Molecular Biology, Wright State University School of Medicine, Dayton, Ohio 45435, USA.
Henkels
K M
KM
Turchi
J J
JJ
D000818:Animals; D001483:Base Sequence; D002417:Cattle; D004247:DNA / Q000096:biosynthesis* / Q000737:chemistry; D004257:DNA Polymerase II / Q000378:metabolism; D019915:DNA Primase; D006367:HeLa Cells; D006801:Humans; D008322:Mammals; D009690:Nucleic Acid Conformation; D012313:RNA / Q000378:metabolism; D012316:RNA Nucleotidyltransferases / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid; D016914:Ribonuclease H; D016615:Telomere / Q000737:chemistry / Q000378:metabolism*; D013950:Thymus Gland / Q000201:enzymology
C034477:RNA primers; D012313:RNA; D004247:DNA; D019915:DNA Primase; D012316:RNA Nucleotidyltransferases; D004257:DNA Polymerase II; D016914:Ribonuclease H; C078856:ribonuclease HI