Rolling circle replication

Rolling circle replication describes a process of unidirectional nucleic acid replication that can rapidly synthesize multiple copies of circular molecules of DNA or RNA, such as plasmids, the genomes of bacteriophages, and the circular RNA genome of viroids. Some eukaryotic viruses also replicate their DNA or RNA via the rolling circle mechanism. Rolling circle replication describes a process of unidirectional nucleic acid replication that can rapidly synthesize multiple copies of circular molecules of DNA or RNA, such as plasmids, the genomes of bacteriophages, and the circular RNA genome of viroids. Some eukaryotic viruses also replicate their DNA or RNA via the rolling circle mechanism. As a simplified version of natural rolling circle replication, an isothermal DNA amplification technique, rolling circle amplification (RCA) was developed. The RCA mechanism is widely used in molecular biology & biomedical nanotechnology, especially in the field of biosensing (as a method of signal amplification). Rolling circle DNA replication is initiated by an initiator protein encoded by the plasmid or bacteriophage DNA, which nicks one strand of the double-stranded, circular DNA molecule at a site called the double-strand origin, or DSO. The initiator protein remains bound to the 5' phosphate end of the nicked strand, and the free 3' hydroxyl end is released to serve as a primer for DNA synthesis by DNA polymerase III. Using the unnicked strand as a template, replication proceeds around the circular DNA molecule, displacing the nicked strand as single-stranded DNA. Displacement of the nicked strand is carried out by a host-encoded helicase called PcrA (the abbreviation standing for plasmid copy reduced) in the presence of the plasmid replication initiation protein. Continued DNA synthesis can produce multiple single-stranded linear copies of the original DNA in a continuous head-to-tail series called a concatemer. These linear copies can be converted to double-stranded circular molecules through the following process: First, the initiator protein makes another nick in the DNA to terminate synthesis of the first (leading) strand. RNA polymerase and DNA polymerase III then replicate the single-stranded origin (SSO) DNA to make another double-stranded circle. DNA polymerase I removes the primer, replacing it with DNA, and DNA ligase joins the ends to make another molecule of double-stranded circular DNA. As a summary, a typical DNA rolling circle replication has five steps: Some DNA viruses replicate their genomic information in host cells via rolling circle replication. For instance, human herpesvirus-6 (HHV-6)(hibv) expresses a set of 'early genes' that are believed to be involved in this process. The long concatemers that result are subsequently cleaved between the pac-1 and pac-2 regions of HHV-6's genome by ribozymes when it is packaged into individual virions. Human Papillomavirus-16 (HPV-16) is another virus that employs rolling replication to produce progeny at a high rate. HPV-16 infects human epithelial cells and has a double stranded circular genome. During replication, at the origin, the E1 hexamer wraps around the single strand DNA and moves in the 3' to 5' direction. In normal bidirectional replication, the two replication proteins will disassociate at time of collision, but in HPV-16 it is believed that the E1 hexamer does not disassociate, hence leading to a continuous rolling replication. It is believed that this replication mechanism of HPV may have physiological implications into the integration of the virus into the host chromosome and eventual progression into cervical cancer. In addition, geminivirus also utilizes rolling circle replication as its replication mechanism. It is a virus that is responsible for destroying many major crops, such as cassava, cotton, legumes, maize, tomato and okra. The virus has a circular, single stranded, DNA that replicates in host plant cells. The entire process is initiated by the geminiviral replication initiator protein, Rep, which is also responsible for altering the host environment to act as part of the replication machinery. Rep is also strikingly similar to most other rolling replication initiator proteins of eubacteria, with the presence of motifs I, II, and III at is N terminus. During the rolling circle replication, the ssDNA of geminivirus is converted to dsDNA and Rep is then attached to the dsDNA at the origin sequence TAATATTAC. After Rep, along with other replication proteins, binds to the dsDNA it forms a stem loop where the DNA is then cleaved at the nanomer sequence causing a displacement of the strand. This displacement allows the replication fork to progress in the 3’ to 5’ direction which ultimately yields a new ssDNA strand and a concatameric DNA strand.