Dataset Information

OriD structure controls RepD initiation during rolling-circle replication.

ABSTRACT: Bacterial antibiotic resistance is often carried by circular DNA plasmids that are copied separately from the genomic DNA and can be passed to other bacteria, spreading the resistance. The chloramphenicol-resistance plasmid pC221 from Staphylococcus aureus is duplicated by a process called asymmetric rolling circle replication. It is not fully understood how the replication process is regulated but its initiation requires a plasmid-encoded protein called RepD that nicks one strand of the parent plasmid at the double-stranded origin of replication (oriD). Using magnetic tweezers to control the DNA linking number we found RepD nicking occurred only when DNA was negatively supercoiled and that binding of a non-nicking mutant (RepDY188F) stabilized secondary structure formation at oriD. Quenched-flow experiments showed the inverted complementary repeat sequence, ICRII, within oriD was most important for rapid nicking of intact plasmids. Our results show that cruciform formation at oriD is an important control for initiation of plasmid replication.

SUBMITTER: Toleikis A

PROVIDER: S-EPMC5775427 | biostudies-literature | 2018 Jan

REPOSITORIES: biostudies-literature

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oriD structure controls RepD initiation during rolling-circle replication.

Toleikis Algirdas A Webb Martin R MR Molloy Justin E JE

Scientific reports 20180119 1

Bacterial antibiotic resistance is often carried by circular DNA plasmids that are copied separately from the genomic DNA and can be passed to other bacteria, spreading the resistance. The chloramphenicol-resistance plasmid pC221 from Staphylococcus aureus is duplicated by a process called asymmetric rolling circle replication. It is not fully understood how the replication process is regulated but its initiation requires a plasmid-encoded protein called RepD that nicks one strand of the parent ...[more]

PMID: 29352198

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Project description:Nucleic acid amplification is a hugely important technology for biology and medicine. While the polymerase chain reaction (PCR) has been highly useful and effective, its reliance on heating and cooling cycles places some constraints on its utility. For example, the heating step of PCR can destroy biological molecules under investigation and heat/cool cycles are not applicable in living systems. Thus, isothermal approaches to DNA and RNA amplification are under widespread study. Perhaps the simplest of these are the rolling circle approaches, including rolling circle amplification (RCA) and rolling circle transcription (RCT). In this strategy, a very small circular oligonucleotide (e.g., 25-100 nucleotides in length) acts as a template for a DNA or an RNA polymerase, producing long repeating product strands that serve as amplified copies of the circle sequence. Here we describe the early developments and studies involving circular oligonucleotides that ultimately led to the burgeoning rolling circle technologies currently under development. This Account starts with our studies on the design of circular oligonucleotides as novel DNA- and RNA-binding motifs. We describe how we developed chemical and biochemical strategies for synthesis of well-defined circular oligonucleotides having defined sequence and open (unpaired) structure, and we outline the unusual ways in which circular DNAs can interact with other nucleic acids. We proceed next to the discovery of DNA and RNA polymerase activity on these very small cyclic DNAs. DNA polymerase "rolling circle" activities were discovered concurrently in our laboratory and that of Andrew Fire. We describe the surprising efficiency of this process even on shockingly small circular DNAs, producing repeating DNAs thousands of nucleotides in length. RNA polymerase activity on circular oligonucleotides was first documented in our group in 1995; especially surprising in this case was the finding that the process occurs efficiently even without promoter sequences in the circle. We describe how one can encode cleavable sites into the product DNAs and RNAs from RCA/RCT, which can then be resolved into large quantities of almost pure oligonucleotides. Our Account then proceeds with a summary describing a broad variety of tools and methods built in many laboratories around the rolling circle concept. Among the important developments are the discovery of highly efficient DNA polymerases for RCA; the invention of exponential ("hyperbranched") RCA amplification made possible by use of a second primer; the development of the "padlock" process for detection of nucleic acids and proteins coupled with RCA; the use of circular oligonucleotides as vectors in cells to encode biologically active RNAs via RCT; and the use of small DNA circles to encode and extend human telomeres. Finally, we finish with some ideas about where the field may go in the future.

Dataset Information

OriD structure controls RepD initiation during rolling-circle replication.

Publications

oriD structure controls RepD initiation during rolling-circle replication.

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