Features in short guanine-rich sequences that stimulate DNA polymerization in vitro.
We have discovered that short guanine-rich oligonucleotides are able to self-associate into higher order structures that stimulate DNA synthesis in vitro without the addition of a conventional template [Ying, J., Bradley, R. K., Jones, L. B., Reddy, M. S., Colbert, D. T., Smalley, R. E., and Hardin, S. H. (1999) Biochemistry 38, 16461-16468]. Our initial analysis indicated the importance of the presence of three contiguous guanines (G) in an oligonucleotide that stimulates DNA polymerization. To gain insight into and to refine sequence requirements for the unexpected DNA synthesis, we analyzed a 231-member guanine-rich octamer library in a fluorescent nucleotide polymerization assay. We observe that, in addition to three contiguous Gs, the presence of a secondary G cluster within the octamer is essential. Furthermore, the location of the primary G cluster in the center of the molecule is most stimulatory. The majority of the octamers that form extended DNA products have a single non-G base separating the primary and secondary G clusters, the identity of which is predominantly thymine (T). Further, a T 5' or 3' of the primary G cluster positively influences the stimulatory function of the oligonucleotide. Overall, the occurrence of bases in the octamer is in the descending order of G > T > A > C. Our studies demonstrate that structures stabilized by noncanonical base pairings are recognized by a DNA polymerase in vitro, and these findings may have relevance within the cell. In particular, the features of these G-rich stimulatory sequences show striking similarities to telomeric sequences that form diverse G-quartet structures in vitro.
Reddy
Mitsu S
MS
Department of Biology and Biochemistry, Institute of Molecular Biology, University of Houston, Houston, Texas 77204-5001, USA.
Hardin
Susan H
SH
D001482:Base Composition; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000096:biosynthesis* / Q000737:chemistry* / Q000145:classification; D003848:Deoxyguanine Nucleotides / Q000737:chemistry; D005456:Fluorescent Dyes; D015723:Gene Library; D006147:Guanine / Q000737:chemistry* / Q000145:classification; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000096:biosynthesis / Q000138:chemical synthesis / Q000145:classification; D011108:Polymers / Q000138:chemical synthesis / Q000737:chemistry*; D012235:Rhodamines; D017422:Sequence Analysis, DNA; D013698:Templates, Genetic
D003848:Deoxyguanine Nucleotides; D005456:Fluorescent Dyes; D009838:Oligodeoxyribonucleotides; D011108:Polymers; D012235:Rhodamines; D006147:Guanine; D004247:DNA