Helicase enzymes

Normally (in vivo) the DNA double helix would be separated by the enzyme helicase, but in PCR (in vitro) the strands are separated by heating to 95 °C for two minutes. This breaks the hydrogen bonds between the two DNA strands. 

The sequence of nucleotides in DNA in can be read only by first separating the DNA double helix (performed by the enzyme helicase). The information is read by RNA polymerase, which then transcribes a corresponding piece of RNA. If the purpose of the RNA is to encode a protein, it can then be read by a structure called a ribosome, which creates proteins according to the specific ordering of the nucleotides in the RNA strand. 

Step 1. Unwinding of the double helix Replication begins when the enzyme helicase catalyzes the separation and unwinding of the nucleic acid strands at a specific point along the DNA helix. In this process, hydrogen bonds between complementary base pairs are broken, and the bases that were formerly in the center of the helix are exposed. The point where this unwinding takes place is called a replication fork (see I Figure 11.11). 

Dicer represents the key enzyme in the RNAi pathway. Dicer is also known as Helicase with RNAse motif, heRNA, Helicase-moi, K12H4.8-like, or KIAA0928. Dicer produces cleaves long double-stranded RNA into small pieces of about 21-23 nucleotides. These so-called siRNA duplexes produced by the action of Dicer contain 5 -phosphates and free 3 -hydroxylgroups... 

The helicases are enzymes central to life itself. The nature of double-stranded DNA means that before a polymerase can begin to copy the appropriate region of the nucleic acid, the two strands have to be unwound the separation of the two strands is the function of the helicase (Fig. 2). An indication of the significance of this family of enzymes is seen in the so-called Werner syndrome, where the helicase function required in the suppression of inappropriate recombination events is defective and causes genomic instability and cancer (for a review see Cobb and Bjergbaek 2006). 

Since the pioneering work of Kleymann et al. (2002), Betz et al. (2002), Baumeister et al. (2007), and Crute et al. (2002), who showed that compounds identified as inhibitors of the helicase-primase enzyme complex could alleviate herpesvirus-induced disease in animal models, the attention of researchers developing antiviral compounds has been drawn more and more towards the virus-encoded helicases, particularly those of Herpes viruses and of RNA viruses such as Hepatitis C Virus (HCV) and SAKS coronavirus (SARS-CoV). Enzyme activity is usually assayed by measuring NTPase activity in the presence of an appropriate nucleic acid co-substrate although, more recently, novel fiuorimetric and luminescence principles have been applied to the measurement of strand unwinding and/or translocation of the protein along the nucleic acid (Frick 2003, 2006). 

Karvinen, J., Laitala, V., Makinen, M. L., Mulari, O., Tamminen, J., Hermonen, J., Hurskainen, P. and Hemmila, I. (2004). Fluorescence quenching-based assays for hydrolyzing enzymes. Application of time-resolved fluorometry in assays for caspase, helicase, and phosphatase. Anal. Chem. 76, 1429-1436. 

An in-depth study of DNA repair systems (Aravind et al., 1999a) has concluded that few, if any, repair proteins occur with identical collinear domain arrangements in all three kingdoms of life. Approximately 10 enzyme families of adenosine triphosphatases (ATPases), photolyases, helicases, and nucleases were identified that are all likely to have been present in the cenancestor. These enzymatic domains are accompanied in DNA repair proteins by numerous regulatory domains. This indicates that the domain architectures of these proteins are labile, with incremental addition and/or subtraction of domains to conserved cores to be a common phenomenon except in the most closely related species. 

Johnson, Daniel S., Lu Bai, Benjamin Y. Smith, Smita S. Patel, and Michelle D. Wang. Single-Molecule Studies Reveal Dynamics of DNA Unwinding by the Ring-Shaped T7 ffelicase. Cell 129 (2007) 1,299-1,309. By using a laser beam, the experimenters made precise measurements of the movement of the bead, observing the forces imposed by helicase enzymes. 

The contraindications to and tolerance of interferon-based therapies in the treatment of HCV infection are similar to those described for patients with HBV infection. As for HBV infection, considerable efforts are being made to develop new agents to improve response rates in patients with HCV infection. Direct antiviral strategies with antisense oligonucleotides, ribozymes, and inhibitors of the viral enzymes— polymerase, helicase, and protease—are under investigation. However, it is likely that interferons will continue to serve as the foundation of therapy for HCV infections, with new agents serving as adjuncts. 

Elongation The elongation phase of replication includes two distinct but related operations leading strand synthesis and lagging strand synthesis. Several enzymes at the replication fork are important to the synthesis of both strands. Parent DNA is first unwound by DNA helicases, and the resulting topological stress is relieved by topo-isomerases. Each separated strand is then stabilized by... 

The process of eukaryotic DNA replication closely follows that of prokaryotic DNA synthesis. Some differences, such as the multiple origins of replication in eukaryotic cells versus single origins of replication in prokaryotes, have already been discussed. Eukaryotic single-stranded DNA-binding proteins and ATP-dependent DNA helicases have been identified, whose functions are analogous to those of the prokaryotic enzymes previously discussed. In contrast, RNA primers are removed by RNase H. 

---