Replication of the circular bacterial chromosome, composed of roughly 5 × 10⁶ bp of DNA proceeds from a single ori. This process is completed in about 30 minutes, a replication rate of 3 × 10⁵ bp/min. The entire mammalian genome replicates in approximately 9 hours, the average period required for formation of a tetraploid genome from a diploid genome in a replicating cell. If a mammalian genome (3 × 10⁹ bp) replicated at the same rate as bacteria (ie, 3 × 10⁵ bp/min) from but a single ori, replication would take over 150 hours! Metazoan organisms get around this problem using two strategies. First, replication is bidirectional. Second, replication proceeds from multiple origins in each chromosome (a total of as many as 100 in humans). Thus, replication occurs in both directions along all of the chromosomes, and both strands are replicated simultaneously. This replication process generates “replication bubbles” (Figure 1).

Fig1. The generation of “replication bubbles” during the process of DNA synthesis. The bidirectional replication and the proposed positions of unwinding proteins at the replication forks are depicted.

The multiple ori sites that serve as origins for DNA replication in eukaryotes are poorly defined except in a few animal viruses and in yeast. However, it is clear that initiation is regulated both spatially and temporally, since clusters of adjacent sites initiate replication synchronously. Replication firing, or DNA replication initiation at a replicator/ori, is influenced by a number of distinct properties of chromatin structure that are just beginning to be understood. It is clear, however, that there are more replicators and excess ORC than needed to replicate the mammalian genome within the time of a typical S phase. Therefore, mechanisms for controlling the excess ORC-bound replicators must exist. Understanding the control of the formation and firing of replication complexes is one of the major challenges in this field.

During the replication of DNA, there must be a separation of the two strands to allow each to serve as a template by hydrogen bonding its nucleotide bases to the incoming deoxynucleoside triphosphate. The separation of the DNA strands is promoted by SSBs in E. coli, and a protein termed replication protein A (RPA) in eukaryotes. These molecules stabilize the single-stranded structure as the replication fork progresses. The stabilizing proteins bind cooperatively and stoichiometrically to the single strands without interfering with the abilities of the nucleotides to serve as templates. In addition to separating the two strands of the double helix, there must be an unwinding of the molecule (once every 10 nucleotide pairs) to allow strand separation. The hexameric dnaB protein complex unwinds DNA in E. coli, whereas the hexameric MCM complex unwinds eukaryotic DNA. This unwinding happens in segments adjacent to the replication bubble. To counteract this unwinding, there are multiple “swivels” interspersed in the DNA molecules of all organisms. The swivel function is provided by specific enzymes that introduce "nicks” in one strand of the unwinding double helix, thereby allowing the unwinding process to proceed. The nicks are quickly resealed without requiring energy input, because of the formation of a high-energy covalent bond between the nicked phosphodiester backbone and the nicking-sealing enzyme. The nicking-resealing enzymes are called DNA topoisomerases. This process is depicted diagrammatically in Figure 2 and there compared with the ATP-dependent resealing carried out by the DNA ligases. Topoisomerases are also capable of unwinding supercoiled DNA. Supercoiled DNA is a higher-ordered structure occurring in circular DNA molecules wrapped around a core, as depicted in Figures 3 and 4.

Fig2. Two types of DNA nick-sealing reactions. Two forms of nick sealing are represented: ATP-independent (top) and ATP dependent (bottom). Nick sealing processes proceeds in multiple steps: (i)-substrate to → (iv)-product. The enzymes involved are signified by E (top, bottom), while small molecule reactants and products are indicated as Phosphate (P); Pyrophosphate (PP), inorganic Phosphate (Pi) generated from PP by the action of ubiquitous pyrophosphatases, Ribose (R), and Adenine (A). The nick-sealing reaction at the top is catalyzed by DNA topoisomerase I and is ATP-energy independent because the energy for reformation of DNA phosphodiester bonds is stored within the covalent attachment of topoisomerase I to DNA (P-E; top; step ii). Bond reformation is accomplished by the nucleophilic attack of the 3′ OH group (green arrow, step iii) to the phosphate of the P-E complex. This reaction releases free topoisomerase I (E) and intact double stranded DNA (step iv). The overall enzyme reaction is schematized at the bottom of the figure (steps i → iv). The nick-sealing reaction catalyzed by DNA ligase (bottom) repairs single strand DNA breaks in the phosphodiester backbone that are a result of DNA replication and/or DNA repair (step i; bottom). The complete DNA ligase reaction requires hydrolysis of two of the high-energy phosphodiester bonds of ATP. The overall reaction scheme of DNA ligase nick-sealing from nick, to enzyme-DNA binding, to enzyme activation that releases Pyrophosphate (PP) to release of free enzyme, AMP and intact DNA is depicted (bottom; as noted in the text, PP is rapidly converted to 2 moles of Pi by the action of ubiquitous pyrophosphatases). The activated ligase (E-P-R-A) reacts with the 5′ P at the nick site to form a transient DNA-P-P-R-A complex (note: P-R-A = AMP) that liberates free DNA Ligase Enzyme (E). Nucleophilic attack of the free 3′ OH group with the 5’P of the DNA-5’P-AMP complex (green arrow, step iii) reseals the nick and liberates AMP. The overall enzyme reaction converting nicked DNA to intact DNA (E + ATP→ E + AMP + 2Pi) is schematized at the bottom of the figure (steps i → iv).

Fig3. Model for the structure of the nucleosome. DNA is wrapped around the surface of a protein cylinder consisting of two each of histones H2A, H2B, H3, and H4 that form the histone octamer. The ~145 bp of DNA, consisting of 1.75 superhelical turns, are in contact with the histone octamer. The position of histone H1, when it is present, is indicated by the dashed outline at the bottom of the figure. Note that histone H1 interacts with DNA as it enters and exits the nucleosome.

Fig4. Supercoiling of DNA.A left-handed toroidal (solenoidal) supercoil, at left, will convert to a right-handed inter wound supercoil, at right, when the cylindric core is removed. Such a transition is analogous to that which occurs when nucleosomes are disrupted by the high salt extraction of histones from chromatin.

There exists in one species of animal viruses (retroviruses) a class of enzymes capable of synthesizing a single-stranded and then a dsDNA molecule from a single-stranded RNA template. This polymerase, termed RNA-dependent DNA polymerase, or “reverse transcriptase,” first synthesizes a DNA–RNA hybrid molecule utilizing the RNA genome as a template. A specific virus-encoded nuclease, RNase H, degrades the hybridized template RNA strand. Subsequently, the remaining DNA strand in turn serves as a template for the viral reverse transcriptase to form a dsDNA molecule containing the genetic information originally present in the RNA genome of the animal virus. The resulting dsDNA can then integrate into the host genome, and from this integrated proviral DNA, viral genes can be expressed via transcription using host cell machinery.

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قسم بين الحرمين يباشر أعمال صيانة الأرصفة في الساحة الوسطية والمناطق المحيطة بها وإدامتها

قسم الشؤون الفكرية ينظم حفل التكليف الشرعي للفتيات في بغداد تيمنًا بذكرى المولد النبوي الشريف

انطلاق النسخة التاسعة من البرنامج التطويري لمشروع (حاملة الرسالة الزينبية)

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دراسة تكشف تأثير "تيك توك" وتطبيقات الفيديو على سلوك الأطفال

"أسرار خفية" في الشاي الأخضر.. تغير قواعد الصحة والوزن

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أول قطار يعمل بالهيدروجين في أميركا يدخل الخدمة رسميًا

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روسيا: تطلق قمرين اصطناعيين بواسطة الصاروخ سويوز 2.1

ميزة ثورية.. أبل تدخل عالم قياس ضغط الدم

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مواضيع ذات صلة

DNA Synthesis Occurs During the S Phase of the Cell Cycle

Initiation & Elongation of DNA Synthesis

The Exact Function of Much of the Mammalian Genome is Not Well Understood

DNA is organized into Chromosomes

Some Regions of Chromatin are “Active” & Others are “Inactive”

The Nucleosome Contains Histone & DNA

Histones Are the Most Abundant Chromatin Proteins

Specific Nucleases Digest Nucleic Acids

Directions for Future Research in Molecular Biology

The ras-fos-jun Pathway

Recessive Oncogenic Mutations, Tumor Suppressors

Small RNA