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Comparisons between chemical mapping and binding to isoenergetic oligonucleotide microarrays reveal unexpected patterns of binding to the Bacillus subtilis RNase P RNA specificity domain.

ABSTRACT: Microarrays with isoenergetic pentamer and hexamer 2'-O-methyl oligonucleotide probes with LNA (locked nucleic acid) and 2,6-diaminopurine substitutions were used to probe the binding sites on the RNase P RNA specificity domain of Bacillus subtilis. Unexpected binding patterns were revealed. Because of their enhanced binding free energies, isoenergetic probes can break short duplexes, merge adjacent loops, and/or induce refolding. This suggests new approaches to the rational design of short oligonucleotide therapeutics but limits the utility of microarrays for providing constraints for RNA structure determination. The microarray results are compared to results from chemical mapping experiments, which do provide constraints. Results from both types of experiments indicate that the RNase P RNA folds similarly in 1 M Na(+) and 10 mM Mg(2+).

SUBMITTER: Liang R

PROVIDER: S-EPMC2938832 | biostudies-literature | 2010 Sep

REPOSITORIES: biostudies-literature

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Comparisons between chemical mapping and binding to isoenergetic oligonucleotide microarrays reveal unexpected patterns of binding to the Bacillus subtilis RNase P RNA specificity domain.

Liang Ruiting R Kierzek Elzbieta E Kierzek Ryszard R Turner Douglas H DH

Biochemistry 20100901 37

Microarrays with isoenergetic pentamer and hexamer 2'-O-methyl oligonucleotide probes with LNA (locked nucleic acid) and 2,6-diaminopurine substitutions were used to probe the binding sites on the RNase P RNA specificity domain of Bacillus subtilis. Unexpected binding patterns were revealed. Because of their enhanced binding free energies, isoenergetic probes can break short duplexes, merge adjacent loops, and/or induce refolding. This suggests new approaches to the rational design of short olig ...[more]

PMID: 20557101

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Project description:RNA-DNA hybrids are common in chromosomal DNA. Persistent RNA-DNA hybrids result in replication fork stress, DNA breaks, and neurological disorders in humans. During replication, Okazaki fragment synthesis relies on frequent RNA primer placement, providing one of the most prominent forms of covalent RNA-DNA strands in vivo The mechanism of Okazaki fragment maturation, which involves RNA removal and subsequent DNA replacement, in bacteria lacking RNase HI remains unclear. In this work, we reconstituted repair of a linear model Okazaki fragment in vitro using purified recombinant enzymes from Bacillus subtilis We showed that RNase HII and HIII are capable of incision on Okazaki fragments in vitro and that both enzymes show mild stimulation by single-stranded DNA binding protein (SSB). We also showed that RNase HIII and DNA polymerase I provide the primary pathway for Okazaki fragment maturation in vitro Furthermore, we found that YpcP is a 5' to 3' nuclease that can act on a wide variety of RNA- and DNA-containing substrates and exhibits preference for degrading RNA in model Okazaki fragments. Together, our data showed that RNase HIII and DNA polymerase I provide the primary pathway for Okazaki fragment maturation, whereas YpcP also contributes to the removal of RNA from an Okazaki fragment in vitroIMPORTANCE All cells are required to resolve the different types of RNA-DNA hybrids that form in vivo When RNA-DNA hybrids persist, cells experience an increase in mutation rate and problems with DNA replication. Okazaki fragment synthesis on the lagging strand requires an RNA primer to begin synthesis of each fragment. The mechanism of RNA removal from Okazaki fragments remains unknown in bacteria that lack RNase HI. We examined Okazaki fragment processing in vitro and found that RNase HIII in conjunction with DNA polymerase I represent the most efficient repair pathway. We also assessed the contribution of YpcP and found that YpcP is a 5' to 3' exonuclease that prefers RNA substrates with activity on Okazaki and flap substrates in vitro.

Dataset Information

Comparisons between chemical mapping and binding to isoenergetic oligonucleotide microarrays reveal unexpected patterns of binding to the Bacillus subtilis RNase P RNA specificity domain.

Publications

Comparisons between chemical mapping and binding to isoenergetic oligonucleotide microarrays reveal unexpected patterns of binding to the Bacillus subtilis RNase P RNA specificity domain.

Similar Datasets

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