Introduction to Molecular Biology
Martin G. Pomper, Juri G. Gelovani, Benjamin Tsui, Kathleen Gabrielson, Richard Wahl, S. Sam Gambhir, Jeff Bulte, Raymond Gibson, William C. Eckelman in Molecular Imaging in Oncology, 2008
Although knowledge of the structure of the replication machinery in eukaryotes is still limited due to its complexity, there are many similarities with the simpler prokaryotes DNA replication process. In eukaryotic cells, DNA exists in the nucleus as a very compact and condensed structure. In order to begin the replication process, this structure must be opened up, so the DNA polymerase enzyme can copy the DNA template. The replication process takes place at a specific site called origin of replication, which is rich in AT content. The first step in DNA replication begins with the binding of the origin recognition complex (ORC) to the origin of replication. ORC complex is a hexamer of related proteins that function as a replication initiation factor that promotes the unwinding or denaturation of DNA. Following the binding of the ORC complex, other proteins (Cdc6/Cdc18 and Cdt1) will bind and coordinate the recruitment of the minichromosome maintenance function (MCM) complex to the origin of replication. The MCM complex is a hexamer and is thought to be the major DNA helicase in eukaryotic organisms. Once the binding of MCM occurs, a fully licensed pre-initiation replication complex (pre-RC) now exists. This process occurs during the G1 phase of the cell cycle and therefore, cannot initiate the replication. Replication only occurs during the S phase. Thus, separating the licensing and activation is a mechanism that ensures only one replication per origin in a cell cycle.
Biology of microbes
Philip A. Geis in Cosmetic Microbiology, 2006
To understand the process in detail requires far more effort. In reality, at least seven enzymes are involved in the process described in the paragraph above: initiator protein, helicase, polymerases, repair nucleases, topoisomerase, single-strand DNA-binding proteins, and DNA ligase. The initiator protein first finds the right place to begin copying and guides the helicase to the correct position (an origin of replication site) on the nucleic acid. The helicase separates the DNA by breaking the weak bonds between the nucleotides to unwind the two strands of DNA. Then the polymerases arrive to join the free nucleotides to their matching complements on the old strands using the phosphate bond energy from the nucleotide to help form the new bond to the other nucleotides as they are added to the existing chain. These polymerases work along with primases that first synthesize a short (one to five nucleotides long) RNA primer. This primer allows DNA polymerase to begin catalyzing the addition of nucleotides to a new strand complementary to the existing template upon which the new DNA synthesis is based.
Affinity Modification in Biochemistry, Biology, and Applied Sciences
Dmitri G. Knorre, Valentin V. Vlassov in Affinity Modification of Biopolymers, 1989
Therefore, RNA polymerases are able to recognize definite regions of double-stranded DNAs, usually called promoters. These regions strongly differ for RNA polymerases of different origin. Promoters used by some definite polymerase bear common features. Thus, all promoters recognized by the most-studied E. coli RNA polymerase have rather similar sequences in the region preceding the transcribed part of the template (Pribnow box) and in the region residing nearly 35 nucleotides prior to the initiation point as shown in Chapter 1, Figure 16. At the same time, DNA polymerase is unable to initiate formation of new chains and needs in a preexisting oligonucleotide (primer) complementary to some region of the template DNA. In experimental conditions this may be achieved by the use of synthetic oligonucleotides. In living systems replication starts by special RNA polymerases called primases which initiate the synthesis of short RNA primers further elongated by DNA polymerase. Primase recognizes definite points at the template DNA which thus are the points of origin of replication. At the final steps of replication short RNA pieces are eliminated by special enzymes.
The clinical use of parvovirus B19 assays: recent advances
Published in Expert Review of Molecular Diagnostics, 2018
Giorgio Gallinella
B19V shares structural features with other viruses in the family (for more extended reviews, see refs [2,3]). The genome is a linear ssDNA molecule of 5.6 kb in length, strands of either polarity are separately encapsidated at the same frequency and are functionally equivalent. The genome organization is composed of a unique internal region, containing all the coding sequences, flanked by repeated, inverted terminal regions that serve as origins of replication. The unique internal region encodes for three major proteins, the non-structural protein NS in the left side, and the two colinear capsid proteins, VP1 and VP2, in the right side, and for additional minor non-structural proteins. The capsid, composed of 5–10% VP1 and 90–95% VP2 proteins, forms an icosahedral structure in T = 1 arrangement, about 25 nm in diameter. VP proteins produced in heterologous expression systems can self-assemble into viral-like particles (VLPs) that are structurally analogous to native virions and are used in diagnostic immunoassays, as well as for a candidate vaccine antigen (Figure 1).
Liver-directed gene-based therapies for inborn errors of metabolism
Published in Expert Opinion on Biological Therapy, 2021
Pasquale Piccolo, Alessandro Rossi, Nicola Brunetti-Pierri
AAV is a small non-enveloped, nonpathogenic virus of the Parvoviridae family. Its single stranded DNA genome contains sequences encoding for viral replication machinery (rep) and capsid (cap) proteins flanked by palindromic inverted terminal repeats (ITR) containing the origin of replication and packaging signals. Naturally occurring AAV are replication defective and they establish latent asymptomatic infections when co-infection with a helper virus (typically adenovirus or herpes simplex virus) occurs. As a result of the infection, anti-AAV antibodies can be found in most subjects, with AAV2 showing the highest prevalence [12]. The complete lack of pathogenicity of AAV has been recently challenged by the finding of clonal integration of AAV genomes into human HCC tissues [13,14].
Determination of copy number and circularization ratio of Tn916-Tn1545 family of conjugative transposons in oral streptococci by droplet digital PCR
Published in Journal of Oral Microbiology, 2019
Tracy Munthali Lunde, Adam P. Roberts, Mohammed Al-Haroni
Four sequenced bacterial strains; B. subtilis BS34A (NZ_LN680001.1), B. subtilis BS49 (NZ_LN649259.1) E. faecium OrEc1, and E. faecium OrEc2 (unpublished data) were used to determine the accuracy of ddPCR in detecting multiple copies of Tn916-Tn1545-like elements. In B. subtilis BS49, E. faecium OrEc1, and E. faecium OrEc2, we were able to accurately detect the expected number of elements using amyE as a chromosomally located, single copy, reference gene. In B. subtilis BS34A however, the ratio between Tn916-Tn1545-like elements (represented by the intTn/xisTn genes) and the reference gene amyE was below one copy (approximately 0.75). The lower ratio may be explained by the chromosomal positioning of the two targets in relation to the origin of replication. In B. subtilis BS34A, the amyE gene (327,604–329,583 bp) is situated closer to the origin of replication in comparison to Tn916 which is in position 1,886,552–1,904,583 bp. The closer proximity of amyE to the origin of replication may result in more targets of the reference gene due to the occurrence of multiple replication forks within a cell prior to cell division, as has been previously reported in B. subtilis [34].
Related Knowledge Centers
- DNA Replication
- DNA Virus
- Genome
- Semiconservative Replication
- Polyploidy
- Cell Cycle
- Aneuploidy
- Chromosome
- Rna Virus
- Double-Stranded Rna Viruses