DNA Strand

Okazaki Fragment

L.J. Reha-Krantz , in Brenner's Encyclopedia of Genetics (Second Edition), 2013

DNA Replication Is Semiconservative

DNA strands are polymers or chains of deoxynucleoside monophosphates that are linked together by phosphodiester bonds ( Figure 1 (a)). The DNA strands have the opposite orientation: one strand is in the 5′ to 3′ direction with respect to the carbon atoms on the sugar (deoxyribose) and the complementary strand is in the 3′ to 5′ direction ( Figure 1 (a)). The two DNA strands are separated during DNA replication and each parental strand serves as a template for the synthesis of a new daughter strand ( Figure 1 (b)). After replication, there will be two double-stranded DNAs; each will have one parental DNA strand and one newly synthesized DNA strand. Because the original double-stranded DNA is not conserved but one parental strand is found in each new duplex DNA, replication is said to be semiconservative. This 'rule' of DNA replication was demonstrated by Meselson and Stahl in 1958.

Figure 1. DNA structure: (a) the chemical structure of double-stranded DNA and (b) semiconservative DNA replication.

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Replication Fork

L.J. Reha-Krantz , L. Zhang , in Brenner's Encyclopedia of Genetics (Second Edition), 2013

DNA Replication Forks Are Sites of Ongoing DNA Replication

DNA strands are polymers or chains of deoxynucleoside monophosphates (dNMPs) that are linked together by phosphodiester bonds ( Figure 1 (a)). The chromosomes of many organisms are composed of two DNA strands: one strand is oriented in the 5′–3′ direction with respect to the carbon atoms on the sugar (deoxyribose) and the complimentary strand is in the opposite 3′–5′ direction. The two DNA strands are held together by hydrogen (H) bonds formed between the bases adenine and thymine to form the AT base pair and between the bases guanine and cytosine to form the GC base pair. Watson and Crick published the double helical structure of DNA in 1953 ( Figure 1 (b)).

Figure 1. DNA structure: (a) the chemical structure of double-stranded DNA, (b) semi-conservative DNA replication, and (c) DNA replication is in the 5′–3′ direction.

Watson and Crick stated that "It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material." As predicted, the two DNA strands are separated during DNA replication and each parental strand serves as a template for the synthesis of a new, complimentary daughter strand ( Figure 1 (b)). DNA polymerases synthesize the daughter strands using the four building blocks of DNA – the deoxynucleoside triphosphates (deoxynucleoside adenosine triphosphate (dATP), deoxynucleoside cytosine triphosphate (dCTP), deoxynucleoside guanine triphosphate (dGTP), and deoxynucleoside thymine triphosphate (dTTP)). An A (adenine) in the template strand directs the incorporation of the T nucleotide (dTMP), T (thymine) templates the incorporation of A (dAMP), G (guanine) templates the incorporation of C (dCMP), and C (cytosine) templates the incorporation of G (dGMP). After replication, there are two double-stranded DNAs; each with one parental DNA strand and one newly synthesized DNA strand ( Figure 1 (c)). Because the original double-stranded DNA is not conserved, but one parental strand is found in each new duplex DNA, replication is said to be semi-conservative. This rule of DNA replication was demonstrated by Meselson and Stahl in 1958.

Another rule of DNA replication is that DNA polymerases replicate DNA in the 5′–3′ direction ( Figure 1 (a)), which means that DNA polymerases at a replication fork must move in opposite directions with respect to their template strands ( Figure 1 (c)); however, replication of both daughter strands is coupled. To explain this topological problem, R. Okazaki proposed and demonstrated that one DNA strand at the replication fork is synthesized continuously while the second strand is synthesized discontinuously in short fragments ( Figure 2 (a)). The continuously synthesized DNA strand is called the 'leading strand' and the discontinuously synthesized strand is called the 'lagging strand'. The short, lagging strand fragments are called 'Okazaki fragments'.

Figure 2. Both daughter DNA strands are replicated at the same time and in the 5′–3′ direction, but leading strand replication is continuous and lagging strand replication is discontinuous (a). The trombone model for lagging strand replication (b).

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Recombination: DNA-Strand Transferases

W.D. Wright , W.-D. Heyer , in Encyclopedia of Biological Chemistry (Second Edition), 2013

Mediators for Presynaptic Filament Formation

DNA-strand transferases are impeded from nucleating filaments when ssDNA-binding proteins saturate ssDNA. Recombination mediators were defined biochemically as proteins that allow filament formation of DNA-strand transferases on ssDNA coated by the cognate ssDNA-binding protein. The prototypical recombination mediator is UvsY protein from bacteriophage T4, which overcomes the block to ssDNA binding imposed by Gp32 SSB protein to allow UvsX filament formation. RecFOR provides this function in E. coli for RecA protein. In eukaryotes, the situation is more complex. S. cerevisiae Rad52, the ortholog of UvsY and RecO proteins, mediates Rad51 filament formation in vitro, but human protein does not. Instead, in eukaryotes containing the BRCA2 protein, the mediator function is occupied by this protein, which was identified as a central tumor suppressor protein for breast and ovarian cancers.

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The Genetic Information (I)

Antonio Blanco , Gustavo Blanco , in Medical Biochemistry, 2017

DNA Replication is Semiconservative

Each DNA strand of a progenitor cell serves as a template for the synthesis of a new complementary polynucleotide chain that is identical to that of the original cell. This process is known as DNA replication. The DNA received by each daughter cell contains one DNA strand that is newly synthesized at replication, and another strand that is directly received from the parental DNA. For this reason, the replication process is referred to as semiconservative (Fig. 21.1). DNA replication takes place before mitosis, during a limited period of the cell cycle, called S phase.

Figure 21.1. DNA replication.

Chains synthesized de novo are shown in white.

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DNA Damage Responses in Atherosclerosis

Kenichi Shimada , ... Moshe Arditi , in Biological DNA Sensor, 2014

DNA Strand Breaks

DNA strand breaks are produced in intermediate events of natural reactions such as the process of V(D)J recombination during lymphocyte development, which is a kind of programmed double-strand break [37,38]. On the other hand, DNA strand breaks can be caused by oxidative DNA damage or by ionizing radiation (e.g. X-rays and gamma rays) as well as drugs like bleomycin [39,40]. These breaks in the DNA backbone can sometimes cause serious genomic instability, carcinogenesis, and cell death. Defective single-strand break repair often results in neurological diseases rather than carcinogenesis or progeria [41]. Since ROS are one of the major causes of the single-strand breaks, and the high level of oxygen consumption in the nervous system makes it more susceptible to defects in single-strand break repair, it makes sense that single-strand breaks may contribute to neurological disorders [42].

Unrepaired double stranded DNA breaks lead to genomic rearrangements, a common and serious problem for all cells and organisms. These double stranded breaks are associated in patients with some progeroid syndromes such as Werner syndrome, ataxia telangiectasia, and Fanconi's anemia [43,44].

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Modes of Action of Antibacterial Agents

David G. Allison , Peter A. Lambert , in Molecular Medical Microbiology (Second Edition), 2015

DNA Replication

The separated DNA strands are kept apart during replication by a specialized protein (Albert's protein) and, with the separated strands as templates, a series of enzymes produce new strands of DNA. An RNA polymerase then forms short primers of RNA on each strand at specific initiator sites, and DNA polymerase III synthesizes and joins short DNA strands onto the RNA primers. DNA polymerase I, which possesses nucleotidase activity, then removes the primers and replaces them with DNA strands. Finally a DNA ligase joins the DNA strands together to produce two daughter chromosomes. The entire process is closely surveyed and regulated by proofreading stages to ensure that each nucleotide is incorporated according to the sequence specified in the template. So far, no therapeutic antimicrobials are available that specifically target the DNA polymerases.

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General Principles

R.J. Preston , J.A. Ross , in Comprehensive Toxicology, 2010

1.16.3.8 Strand Breakage

DNA strand breaks represent an important type of DNA damage induced by some chemicals and by ionizing radiation. The chemotherapeutic agent bleomycin is an efficient inducer of both single-strand breaks (SSB) and double-strand breaks (DSB). Bleomycin intercalates into the DNA helix and abstracts a hydrogen from C4′ of deoxyribose, inducing a radical capable of leading to either a strand break or an abasic site. These strand breaks are not directly repairable by DNA ligase, rather, several DNA bases must be removed and the sequence resynthesized before the break can be ligated. Many chemicals that induce reactive oxygen species have been shown also to induce SSB. DSB are produced almost exclusively by the radiomimetic enediyne C-1027, which is extremely cytotoxic ( Kennedy et al. 2007). Both SSB and DSB induction can lead to the formation of chromosomal alterations.

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Microbiology of Atypical Environments

Hirak Ranjan Dash , Surajit Das , in Methods in Microbiology, 2018

3.2.2.1 DGGE/temperature gradient gel electrophoresis (TGGE)

DNA strands of the same length and different sequences can be separated by the techniques of DGGE and/or TGGE. In this technique, the whole DNA is extracted from the atypical environmental samples followed by amplification of 16S/18S rRNA gene by PCR and separation of the amplified sequences on a linear gradient of DNA denaturants or temperature ( Strathdee & Free, 2013). Thus, the separation of DNA fragments is based on the differential mobility of the partially single-stranded DNA molecules in an acrylamide gel containing urea and formamide as denaturants (Mühling, Woolven-Allen, Murrell, & Joint, 2008). Additionally, TGGE also employs the same principle, where a gradient of temperature replaces the chemical denaturants in the polyacrylamide gel (Viglasky, 2013). In both the techniques, a GC clamp of 30–50 nucleotides long is attached to the 5′ end of the amplicon which is essential to prevent complete dissociation of the DNA fragment with increase in denaturant concentration or temperature gradients.

Applications of DGGE and TGGE are quite common to diversity studies in atypical environments. Stabilization of saline nitrogen waste water by highly diverse denitrifying bacteria has been well established by DGGE of PCR amplified 16S rRNA gene fragments where a taxonomic affiliation of the dominant microbial species has been established as γ-Proteobacteria (Yoshie et al., 2001). DGGE analysis also revealed the occurrence of Archaea in mangrove trees such as Rhizophora mangle and Laguncularia racemosa in complex atypical environmental conditions (Pires et al., 2012). Application of the DGGE technique further revealed the occurrence of around 15 different prokaryotic taxa belonging to genera Alcanivorax, Pseudoxanthomonas, Bosea, Halomonas and Marinobacter in oil contaminated desert soil, sea water and hypersaline coastal soil (Al-Mailem, Kansour, & Radwan, 2017).

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Noncoding RNAs in Genome Integrity

I. Kovalchuk , in Genome Stability, 2016

5.4.1 Regulation of Sensors

DNA strand breaks are sensed by several groups of proteins, such as the Mre11–Rad50–Nbs1 (MRN) complex, Ku70/80, and 53BP1. Proteins like ATM and γH2AX (the phosphorylated form of H2AX protein) also play an essential role in the initial damage recognition and signaling because H2AX is one of the first immediate targets of ATM phosphorylation. The repair choice is influenced by this initial binding (see Chapter 14). Therefore, the regulation of the abundance of one or several components of these sensors may significantly influence DNA-repair choice and outcomes.

Two component proteins involved in sensing strand breaks, Nbs1 and Ku80, may likely be regulated by miRNAs as they both contain the long 3′-UTRs with a high number of miRNA binding sites that can serve as a potential target for translation inhibition. Indeed, a 2015 work showed that Ku80 expression could indeed be affected by hsa–miR–526b in nonsmall-cell lung carcinoma (NSCLC) [21]. Hsa–miR–526b was found to be downregulated and Ku80 upregulated in the NSCLC cells compared to healthy tissues. No experimental data exist for Nbs1, but an association study demonstrated that NBS1 as well as Mre11 were likely to be regulated by miRNA; a case–control study revealed the association between the presence of SNPs in binding of several miRNAs at the 3′-UTR of these genes with an increased risk of breast cancer development [22]. Similar data for Nbs1 were observed in case-control studies involving colorectal cancer [23].

The expression of ATM is also regulated by miRNA at the posttranslational level; in neuroblastoma and HeLa cells, miR-421 downregulates ATM activity by modulating cell-cycle checkpoints and changing cell sensitivity to IR [24]. Similarly, miR-100 [25], miR-101 [26], and miR-18a [27] are also likely to regulate ATM because all of them were shown to target the 3′-UTR of ATM and downregulate it. Details of miRNA impact on various steps of DSB repair are shown in Fig. 25.1C [15].

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Mycotoxins

Carina Ladeira , in Environmental Mycology in Public Health, 2016

Formamidopyrimidine DNA Glycosylase

Measuring DNA strand breaks gives limited information. Breaks may represent the direct effect of some damaging agent, but generally they are quickly rejoined. They may in fact be apurinic/apyrimidinic (AP) sites baseless sugars, which are alkali labile and therefore appear as breaks. Or they may be intermediates in cellular repair because both nucleotide and base excision–repair processes cut out damage and replace it with sound nucleotides. 62,66 AP sites are alkali labile, so in principle they are expected to appear among the strand breaks, detected in the standard alkaline comet assay. However, it has not been convincingly demonstrated that all AP sites are converted under these conditions. 56,64

To make the assay more specific and sensitive, an extra step was introduced of digesting the nucleoids with an enzyme that recognizes a particular kind of damage and creates a break. FPG detects the major purine oxidation product 8-OHG as well as other altered purines. 55,60,63,65 This enzyme was named for its ability to recognize imidazole-ring–opened purines, or formamidopyrimidines: namely, 8-oxo-G, 2,6-diamino-4-hydroxy-5-formadopyrimidine and 4,6-diamino-5-formamidopyrimidine, which occur during the spontaneous breakdown of damaged purines; however, a major substrate in cellular DNA is 8-OHG. 56,59,61,66

A mammalian analogue of FPG, OGG1, has been applied in the Comet assay; however, studies performed comparing FPG and OGG1 revealed the ineffectiveness of OGG1. 56 For that reason, FPG continues to be the enzyme of choice for oxidized purines.

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