Biofelsefe — DNA Eşlenimi
NFA 2020 / Aziz Yardımlı

 

SİTELER

Biofelsefe — DNA Eşlenimi (Replication)


SİTE İÇİ ARAMA       
 
 
   
 

🛑 EŞLENİM

  • DNA eşlenimi tek bir kökensel DNA molekülünden iki özdeş DNA eşlemi üretme sürecidir.
  • DNA eşlenimi tüm dirimli hücrelerde olur ve kalıtım için özseldir.
  • DNA iki tümleyici telden yapılı bir çifte sarmaldır (bazların ‘tümleyiciliği’ (adenin ↔ thimin) ve (guanin ↔ sitozin) eşlenmesini anlatır).
  • Eşlenim sırasında DNAnın iki teli ayrılır ve her biri yeni tellerin üretimi için kalıp olur.
  • Yeni sarmal bir kökensel tel ve bir de yeni telden oluşacaktır (yarı-tutucu eşlenim).
  • Baz-çiftlemesi DNA eşleminin ve DNA onarımının temelinde yatar.



Yarı-tutucu eşlenim.

  • Hücrenin düzeltme düzenekleri eşlemin hemen hemen tam olarak doğru yapılmasını sağlar.
  • DNAnın eşlenmesi genom üzerindeki belirli başlangıç noktalarında başlar.
  • Helikaz DNAnın tellerinin ayrılmasını sağlar.
  • Polimeraz tümleyici nükleotidleri ekleyerek yeni tellerin bireşimini sağlar.


Ökaryotlarda DNA eşlenimi.
 
DNA EŞLENİMİ VE DNA ONARIMI BAZ-ÇİFTLENMESİ TEMELİNDE OLUR
  • DNA eşleniminde hücre bir DNA telinin nükleotid dizisini yeni bir tümleyici DNA dizisine eşlemede DNA kalıp düzeneğini kullanır.
  • Bu süreç DNA sarmalının iki kalıp teline ayırılmasını gerektirir.
  • Bunu izleyen adım DNA kalıp tellerindeki her bir nükleotidin serbest (polimerize olmamış) bir tümleyici nükleotid tarafından tanınmasıdır.
  • DNA sarmalının ayırılması her bir DNA bazı üzerindeki hidrojen bağlı verici ve alıcı grupları baz-çiftlenmesi için açık bırakır.
  • Gelen nükleotidlerin yeni bir DNA zincirine polimerize edilmesi enzim katalizi yoluyla olur.
  • İlk nükleotid-polimerizasyon enzimi 1957’de keşfedildi.
  • Bu enzimin kullandığı serbest nükleotidler deoksiribonükleosid trifosfatlardır.



DNA çift sarmalındaki tümleyici baz çiftleri.
Bazların şekilleri ve kimyasal yapıları hidrojen bağlarının yalnızca A ve T arasında ve G ve C arasında etkili olarak oluşmasına izin verir, çünkü hidrojen bağları oluşturabilecek atomlar o zaman çifte sarmalı çarpıtmaksızın biraraya getirilebilir. Belirtildiği gibi, A ve T arasında iki hidrojen bağı oluşurken, G ve C arasında üç bağ oluşur. Bazlar bu yolda ancak onları kapsayan iki polinükleotid zinciri birbirine anti-paralel ise çiftlenebilir.



DNA çifte sarmalı kendi eşlenmesi için kalıp olarak hizmet eder.
Nükleotid A ancak T ile ve G ancak C ile çiftlenebileceği için, her bir DNA teli kendi tümleyici telindeki nükleotidler dizisini DNA baz-çiflenmesi ile belirlemek için kalıp olarak hizmet edebilir. Bu yolda bir çift sarmallı DNA molekülü sağın olarak eşlemlenebilir.
 

DNA EŞLENİM ÇATALI BAKIŞIMSIZDIR

  • Bir hücre içideki DNA eşlenimı sırasında iki kökensel DNAdan her biri bütünüyle yeni bir telin oluşumu için bir kalıp olarak hizmet eder.
  • Bölünen bir hücrenin  yeni kuşak DNAlarından her biri bir kökensel ve bir yeni tel kapsayan yeni bir DNA çifte sarmalını kalıt aldığı için,  DNA çifte sarmalının “yarı-tutucu” olarak eşlemlendiği söylenir.


DNA bireşiminin kimyası. Oluş sürecindeki DNA molekülleri.
Bir deoksiribonükleotidin bir polinükleotid zincirinin 3' ucuna (birincil tel) eklenmesi DNA bireşimindeki temel tepkimedir. Gelen bir deoksiribonükleosid ve varolan bir DNA teli (kalıp teli) arasındaki baz-çiftlenmesi yeni DNA telinin oluşumunu belirler ve bir tümleyici nükleotid dizisi kazanmasını sağlar.


DNA bireşimi DNA polimeraz tarafından katalize edilir.

(A) DNA polimeraz bir polinükleotid zincirinin 3’—OH ucuna, varolan bir kalıp teli ile eşlenerek büyümekte olan birincil tele bir deoksiribonükleotidin eklenmesini adım adım katalize eder.  Yeni bireştirilen DNA teli buna göre 5’—3’ yönünde polimerize olur. Gelen her bir deoksiribonükleosidin DNA polimeraz tarafından tanınmak için kalıp teli ile eşlenmesi gerektiğinden, bu tel olanaklı dört deoksiribonükleotidden (A, C, G ya da T) hangisinin ekleneceğini belirler. Tepkime pirofosfatın salınması ve daha sonra iki inorganik fosfat molekülüne hidrolize olması ile ortaya çıkan büyük ve uygun bir serbest-enerji değişimi yoluyla yerine gelir.

(B) DNA polimeraz ve DNA (kavuniçi) karmaşık yapısı X-ışını kristalografisi ile belirlenir. Kalıp DNA teli uzun olan ve yeni bireşmiş DNA kısa olan teldir.

(C) DNA polimerazın B’deki yapı üzerine dayanan şematik çizgesi. Doğru olarak gelen deoksiribonükleosid trifosfatın uygun baz-çifti geometrisi polimerazın baz çifti çevresine sıkıca sarılmasını ve böylelikle nükleotid ekleme tepkimesini başlatmasını sağlar (orta çizge (C)). Pirofosfatın kopması polimerazı gevşeterek DNAnın tek bir nükleotid kadar yer değiştirmesine izin verir ve bununla polimerazın etkin sitesi sonraki deoksiribonükleosidi almaya hazırlanır.



DNA eşleniminin yarı-tutucu doğası.
Bir eşlenim turunda iki DNA telinden her biri tümleyici DNA telinin oluşumu için kalıp olarak kullanılır. Buna göre kökensel teller birçok hücre kuşağı boyunca dokunulmadan kalır.

  • Eşlemleme sırasında, ebeveyn DNA çifte sarmalı boyunca ilerleyen yerel bir eşlemleme bölgesi uzanır.
  • Y-şekilli yapısından ötürü, bu etkin bölgeye bir eşlenim çatalı denir.
  • Eşlenim çatalında DNA polimerazı kapsayan bir çoklu-enzim karmaşası her iki yeni kuşak telin DNAsını bireştirir.
  • DNA çifte sarmalındaki iki DNA telinin anti-paralel yönelimi nedeniyle, polimerizasyon  bir telde 5ʹ—3ʹ yönünde ve ötekinde 3ʹ—5ʹ yönünde ilerler.
  • Eşlenim çatalındaki DNA polimeraz yalnızca 5ʹ—3ʹ yönünde kesintisiz olarak bireşim yapabilir.
  • 3ʹ—5ʹ yönünde bireşimin nasıl yer aldığı 1960’ların sonlarında bulundu.
  • Helikaz tarafından yerine getirilen ayırılmadan sonra, eşlemleme süreci 5ʹ—3ʹ yönelimli telde önden (önceleyen tel) ve 3ʹ—5ʹ yönelimli telde arkadan gelir (geciken tel).
  • Geciken tel kesintisiz olarak üretilemez, çükü 3ʹ—5ʹ yöneliminden ötürü polimeraz geriye doğru ve kesintili olarak çalışmak zorundadır.
  • Kesintili olarak bireşimi yapılan kısa DNA nükleotid tümceleri (Okozaki fragmanları) ökaryotlarda yaklaşık 150-200 baz çifti uzunluğundadır.
  • Okozaki fragmanları DNA ligaz tarafından geciken teli üretmek üzere bağlanır.
  • Tüm eşlemleme süreci bu kesilmelerden ötürü “yarı-sürekli” olarak adlandırılır.
 

 



📹 DNA Replication — Leading Strand vs Lagging Strand & Okazaki Fragments / The Organic Chemistry Tutor (VİDEO)

📹 DNA Replication — Leading Strand vs Lagging Strand & Okazaki Fragments / The Organic Chemistry Tutor (LINK)

This biology video tutorial provides a basic introduction into DNA replication. It discusses the difference between the leading strand and the lagging strand as well as the presence of okazaki fragments. Here is a list of topics:
0:00
- Intro to DNA Replication
0:00
- Semiconservative Replication
0:49
- DNA strands are antiparallel
1:07
- Complementary Base Pairing In DNA
2:05
- Hydrogen Bonds Between Adenine, Thymine, Cytosine, and Guanine In DNA
2:39
- Bidirectionality of DNA and Origin of Replication
3:26
- DNA Helicase and Topoisomerase
4:30 - Single Stranded Binding (SSB) Proteins
4:53 - RNA Primers and Primase
5:27 - DNA Polymerase III
5:49
- Semidiscontinuous Nature of DNA Replication
6:40 - Leading Strand and Lagging Strand
9:00
- Okazaki Fragments
9:13 - The Function of DNA Ligase
9:30 - Exonuclease Activity of DNA Polymerase I and III - Proofreading Ability and DNA Repair

 



📹📹📹 DNA REPLICATION (VİDEO)

📹 Mechanism of DNA Replication (Basic) / DNA Learning Center (VİDEO)

📹 Mechanism of DNA Replication (Basic) / DNA Learning Center (LINK)

Knowing the structure of DNA, scientists speculated and then proved that DNA is the template for copying the genetic code. See how information in DNA is copied to make new DNA molecules. Originally created for DNA Interactive

TRANSCRIPT: Using computer animation based on molecular research, we are now able to see how DNA is actually copied in living cells. You are looking at an assembly line of amazing miniature biochemical machines that are pulling apart the DNA double helix and cranking out a copy of each strand. The DNA to be copied enters the production line from bottom left. The whirling blue molecular machine is called helicase. It spins the DNA as fast as a jet engine as it unwinds the double helix into two strands. One strand is copied continuously and can be seen spooling off to the right. Things are not so simple for the other strand because it must be copied backwards. It is drawn out repeatedly in loops, and copied one section at a time. The end result is two new DNA molecules.

 



📹 Mechanism of DNA Replication (Advanced) / DNA Learning Center (VİDEO)

📹 Mechanism of DNA Replication (Advanced) / DNA Learning Center (LINK)

Knowing the structure of DNA, scientists speculated and then proved that DNA is the template for copying the genetic code. See how information in DNA is copied to make new DNA molecules. Originally created for DNA Interactive ).

TRANSCRIPT: During DNA replication, both strands of the double helix act as templates for the formation of new DNA molecules. Copying occurs at a localized region called the replication fork, which is a Y shaped structure where new DNA strands are synthesised by a multi-enzyme complex. Here the DNA to be copied enters the complex from the left. One new strand is leaving at the top of frame and the other new strand is leaving at bottom. The first step in DNA replication is the separation of the two strands by an enzyme called helicase. This spins the incoming DNA to unravel it: at ten thousand RPM in the case of bacterial systems. The separated strands are called three prime and five prime, distinguished by the direction in which their component nucleotides join up. . The 3' DNA strand, also known as the leading strand, is diverted to a DNA polymerase and is used as a continuous template for the synthesis of the first daughter DNA helix. The other half of the DNA double helix, known as the lagging strand, has the opposite 3' to 5' orientation and consequently requires a more complicated copying mechanism. As it emerges from the helicase, the lagging strand is organised into sections called Okazaki fragments. These are then presented to a second DNA polymerase enzyme in the preferred 5' to 3' orientation. These sections are then effectively synthesised backwards. When the copying is complete, the finished section is released and the next loop is drawn back for replication. Intricate as this mechanism appears, numerous components have been deliberately left out to avoid complete confusion. The exposed strands of single DNA are covered by protective binding proteins. And in some systems, multiple Okazaki fragments may be present. The molecular reality is very different from the iconic image of the double helix neatly separating into two DNA copies as so often depicted.

 



📹 DNA Replication (Updated) / Amobea Sisters (VİDEO)

📹 DNA Replication (Updated) / Amobea Sisters (LINK)

Explore the steps of DNA replication, the enzymes involved, and the difference between the leading and lagging strand! This video is an update from our old DNA replication video with nearly the same script but added detail and improved art!

Table of Contents:
00:00
Intro
0:11
Why do you need DNA replication?
0:27 Where and when?
1:12
Introducing key player enzymes
2:48
Initial steps of DNA Replication
4:04
Explaining 5' to 3' and 3' to 5'
5:09
Showing leading and lagging strands in DNA replication

 



📹 DNA Replication / Bozeman Science (VİDEO)

📹 DNA Replication / Bozeman Science (LINK)

Paul Andersen explains how DNA replication ensures that each cell formed during the cell cycle has an exact copy of the DNA. He describes the Meselson-Stahl experiment and how it showed that DNA copies itself through a semi-conservative process. He then explains how multiple enzymes, like DNA polymerase, helicase, primase, ligase, and single strand binding proteins copy DNA. He also differentiates between the leading and the lagging strand. He explains how DNA is anti-parallel in nature and how eukaryotic cells have multiple origins of replication.

 



📹 DNA Replication Animation / Super EASY (VİDEO)

📹 DNA Replication Animation / Super EASY (LINK)

DNA Replication Animation

 



📹 DNA replication - 3D (VİDEO)

📹 DNA replication - 3D (LINK)

This 3D animation shows you how DNA is copied in a cell. It shows how both strands of the DNA helix are unzipped and copied to produce two identical DNA molecules.

 



📹 A Extracting DNA from fruit / yourgenome (VİDEO)

📹 Extracting DNA from fruit / yourgenome (LINK)

This video shows how to extract DNA from strawberries using everyday items.

 



📹 DNA Sequencing — 3D / yourgenome (VİDEO)

📹 DNA Sequencing — 3D / yourgenome (LINK)

This 3D animation shows the basic steps in the method of DNA sequencing that was used during the Human Genome Project.

 



📹 How the Human Genome was sequenced? / yourgenome (VİDEO)

📹 How the Human Genome was sequenced? / yourgenome (LINK)

The Human Genome Project (HGP) was an immense international enterprise: probably the biggest biological experiment yet attempted. Researchers worked together to read the entire sequence of the DNA letters (bases) in the human genome - more than 3,000,000,000 of them. The 'gold standard' sequence was announced in 2003 and published in 2004.

 



📹 Leading and lagging strands in DNA replication / Khan Academy (VİDEO)

📹 Leading and lagging strands in DNA replication / Khan Academy (LINK)

Roles of DNA polymerase, primase, ligase, helicase and topoisomerase in DNA replication. An explanation of leading and lagging strands.

 



 

 



📹📹📹 DNA REPLICATION (OXFORD) (VİDEO)

📹 01 DNA Replication / Oxford Academic (Oxford University Press) (VİDEO)

📹 01 DNA Replication / Oxford Academic (Oxford University Press) (LINK)

In this animation, we focus on bacteria and explore how they replicate their DNA.

 



📹 02 Replication fork coupling / Oxford Academic (Oxford University Press) (VİDEO)

📹 02 Replication fork coupling / Oxford Academic (Oxford University Press) (LINK)

In this animation, we consider how bacteria achieve the feat of coupling of DNA replication at the replication fork as the lagging and leading strand and synchronously copied.

 



📹 03 Mitosis and Cell Division / Oxford Academic (Oxford University Press) (VİDEO)

📹 03 Mitosis and Cell Division / Oxford Academic (Oxford University Press) (LINK)

In this animation, we explore mitosis, the key process in cell division during which duplicated chromosomes are segregated into separate daughter cells.

 



 

 



  DNA replication (W)

DNA replication (W)

DNA replication (W)


DNA replication: The double helix is un'zipped' and unwound, then each separated strand (turquoise) acts as a template for replicating a new partner strand (green). Nucleotides (bases) are matched to synthesize the new partner strands into two new double helices.

 
   

In molecular biology, DNA replication is the biological process of producing two identical replicas of DNA from one original DNA molecule. DNA replication occurs in all living organisms acting as the most essential part for biological inheritance. The cell possesses the distinctive property of division, which makes replication of DNA essential.

DNA is made up of a double helix of two complementary strands. During replication, these strands are separated. Each strand of the original DNA molecule then serves as a template for the production of its counterpart, a process referred to as semiconservative replication. As a result of semi-conservative replication, the new helix will be composed of an original DNA strand as well as a newly synthesized strand. Cellular proofreading and error-checking mechanisms ensure near perfect fidelity for DNA replication.

In a cell, DNA replication begins at specific locations, or origins of replication, in the genome. Unwinding of DNA at the origin and synthesis of new strands, accommodated by an enzyme known as helicase, results in replication forks growing bi-directionally from the origin. A number of proteins are associated with the replication fork to help in the initiation and continuation of DNA synthesis. Most prominently, DNA polymerase synthesizes the new strands by adding nucleotides that complement each (template) strand. DNA replication occurs during the S-stage of interphase.

DNA replication (DNA amplification) can also be performed in vitro (artificially, outside a cell). DNA polymerases isolated from cells and artificial DNA primers can be used to start DNA synthesis at known sequences in a template DNA molecule. Polymerase chain reaction (PCR), ligase chain reaction (LCR), and transcription-mediated amplification (TMA) are examples.

 
 
DNA Structure

DNA structure

DNA structure (W)

DNA exists as a double-stranded structure, with both strands coiled together to form the characteristic double-helix. Each single strand of DNA is a chain of four types of nucleotides. Nucleotides in DNA contain a deoxyribose sugar, a phosphate, and a nucleobase. The four types of nucleotide correspond to the four nucleobases adenine, cytosine, guanine, and thymine, commonly abbreviated as A, C, G and T. Adenine and guanine are purine bases, while cytosine and thymine are pyrimidines. These nucleotides form phosphodiester bonds, creating the phosphate-deoxyribose backbone of the DNA double helix with the nucleobases pointing inward (i.e., toward the opposing strand). Nucleobases are matched between strands through hydrogen bonds to form base pairs. Adenine pairs with thymine (two hydrogen bonds), and guanine pairs with cytosine (three hydrogen bonds).

DNA strands have a directionality, and the different ends of a single strand are called the "3′ (three-prime) end" and the "5′ (five-prime) end". By convention, if the base sequence of a single strand of DNA is given, the left end of the sequence is the 5′ end, while the right end of the sequence is the 3′ end. The strands of the double helix are anti-parallel with one being 5′ to 3′, and the opposite strand 3′ to 5′. These terms refer to the carbon atom in deoxyribose to which the next phosphate in the chain attaches. Directionality has consequences in DNA synthesis, because DNA polymerase can synthesize DNA in only one direction by adding nucleotides to the 3′ end of a DNA strand.

The pairing of complementary bases in DNA (through hydrogen bonding) means that the information contained within each strand is redundant. Phosphodiester (intra-strand) bonds are stronger than hydrogen (inter-strand) bonds. This allows the strands to be separated from one another. The nucleotides on a single strand can therefore be used to reconstruct nucleotides on a newly synthesized partner strand.

 



 
DNA polymerase

DNA polymerase

DNA polymerase (W)

Main article: DNA polymerase

DNA polymerases are a family of enzymes that carry out all forms of DNA replication. DNA polymerases in general cannot initiate synthesis of new strands, but can only extend an existing DNA or RNA strand paired with a template strand. To begin synthesis, a short fragment of RNA, called a primer, must be created and paired with the template DNA strand.



DNA polymerases adds nucleotides to the 3′ end of a strand of DNA. If a mismatch is accidentally incorporated, the polymerase is inhibited from further extension. Proofreading removes the mismatched nucleotide and extension continues.
 
   

DNA polymerase adds a new strand of DNA by extending the 3′ end of an existing nucleotide chain, adding new nucleotides matched to the template strand one at a time via the creation of phosphodiester bonds. The energy for this process of DNA polymerization comes from hydrolysis of the high-energy phosphate (phosphoanhydride) bonds between the three phosphates attached to each unincorporated base. Free bases with their attached phosphate groups are called nucleotides; in particular, bases with three attached phosphate groups are called nucleoside triphosphates. When a nucleotide is being added to a growing DNA strand, the formation of a phosphodiester bond between the proximal phosphate of the nucleotide to the growing chain is accompanied by hydrolysis of a high-energy phosphate bond with release of the two distal phosphates as a pyrophosphate. Enzymatic hydrolysis of the resulting pyrophosphate into inorganic phosphate consumes a second high-energy phosphate bond and renders the reaction effectively rreversible.[Note 1]

(Note 1: The energetics of this process may also help explain the directionality of synthesis—if DNA were synthesized in the 3′ to 5′ direction, the energy for the process would come from the 5′ end of the growing strand rather than from free nucleotides. The problem is that if the high energy triphosphates were on the growing strand and not on the free nucleotides, proof-reading by removing a mismatched terminal nucleotide would be problematic: Once a nucleotide is added, the triphosphate is lost and a single phosphate remains on the backbone between the new nucleotide and the rest of the strand. If the added nucleotide were mismatched, removal would result in a DNA strand terminated by a monophosphate at the end of the "growing strand" rather than a high energy triphosphate. So strand would be stuck and wouldn't be able to grow anymore. In actuality, the high energy triphosphates hydrolyzed at each step originate from the free nucleotides, not the polymerized strand, so this issue does not exist.)

In general, DNA polymerases are highly accurate, with an intrinsic error rate of less than one mistake for every 107 nucleotides added. In addition, some DNA polymerases also have proofreading ability; they can remove nucleotides from the end of a growing strand in order to correct mismatched bases. Finally, post-replication mismatch repair mechanisms monitor the DNA for errors, being capable of distinguishing mismatches in the newly synthesized DNA strand from the original strand sequence. Together, these three discrimination steps enable replication fidelity of less than one mistake for every 109 nucleotides added.

The rate of DNA replication in a living cell was first measured as the rate of phage T4 DNA elongation in phage-infected E. coli. During the period of exponential DNA increase at 37 °C, the rate was 749 nucleotides per second. The mutation rate per base pair per replication during phage T4 DNA synthesis is 1.7 per 108.

 



 
Replication process

Replication process

Replication process (W)


DNA replication, like all biological polymerization processes, proceeds in three enzymatically catalyzed and coordinated steps: initiation, elongation and termination.

 



Initiation

Initiation (W)


For a cell to divide, it must first replicate its DNA. DNA replication is an all-or-none process; once replication begins, it proceeds to completion. Once replication is complete, it does not occur again in the same cell cycle. This is made possible by the division of initiation of the pre-replication complex.

Pre-replication complex

In late mitosis and early G1 phase, a large complex of initiator proteins assembles into the pre-replication complex at particular points in the DNA, known as "origins". In E. coli the primary initiator protein is DnaA; in yeast, this is the origin recognition complex. Sequences used by initiator proteins tend to be "AT-rich" (rich in adenine and thymine bases), because A-T base pairs have two hydrogen bonds (rather than the three formed in a C-G pair) and thus are easier to strand-separate. In eukaryotes, the origin recognition complex catalyzes the assembly of initiator proteins into the pre-replication complex. Cdc6 and Cdt1 then associate with the bound origin recognition complex at the origin in order to form a larger complex necessary to load the Mcm complex onto the DNA. The Mcm complex is the helicase that will unravel the DNA helix at the replication origins and replication forks in eukaryotes. The Mcm complex is recruited at late G1 phase and loaded by the ORC-Cdc6-Cdt1 complex onto the DNA via ATP-dependent protein remodeling. The loading of the Mcm complex onto the origin DNA marks the completion of pre-replication complex formation.




Overview of the steps in DNA replication.
Duplication of the DNA begins with origin unwinding, followed by the synthesis of RNA primers (jagged lines) on both DNA strands. DNA polymerase delta or epsilon extends these primers by adding new DNA (green lines) only in a 5' to 3' direction. On the leading strands, this results in the continuous synthesis of long DNA molecules. Lagging strands, in contrast, are synthesized discontinuously: primers are placed on the template every ~200 nucleotides and extended to form short Okazaki fragments. For simplicity, this diagram does not show the replacement of primers with DNA or the synthesis of telomeres at the chromosome ends.

 

If environmental conditions are right in late G1 phase, the G1 and G1/S cyclin-Cdk complexes are activated, which stimulate expression of genes that encode components of the DNA synthetic machinery. G1/S-Cdk activation also promotes the expression and activation of S-Cdk complexes, which may play a role in activating replication origins depending on species and cell type. Control of these Cdks vary depending cell type and stage of development. This regulation is best understood in budding yeast, where the S cyclins Clb5 and Clb6 are primarily responsible for DNA replication. Clb5,6-Cdk1 complexes directly trigger the activation of replication origins and are therefore required throughout S phase to directly activate each origin.

In a similar manner, Cdc7 is also required through S phase to activate replication origins. Cdc7 is not active throughout the cell cycle, and its activation is strictly timed to avoid premature initiation of DNA replication. In late G1, Cdc7 activity rises abruptly as a result of association with the regulatory subunit Dbf4, which binds Cdc7 directly and promotes its protein kinase activity. Cdc7 has been found to be a rate-limiting regulator of origin activity. Together, the G1/S-Cdks and/or S-Cdks and Cdc7 collaborate to directly activate the replication origins, leading to initiation of DNA synthesis.




Steps in DNA synthesis.
his diagram shows the synthesis of an Okazaki fragment on the lagging strand; synthesis on the leading strand is not shown but involves the same steps. (a) Helicase unwinds the DNA helix, and the resulting single-stranded DNA is coated with RPA proteins. (b) The polymerase alpha–primase synthesizes a short RNA–DNA primer in the 5' to 3' direction, displacing RPA molecules as it travels along the template. (c) Following completion of the primer, the clamp loader displaces polymerase a and catalyzes loading of the sliding clamp and DNA polymerase delta or epsilon. (d) The polymerase machine extends the primer until it reaches the 5' end of the previous Okazaki fragment. (e) The primer of the downstream Okazaki fragment is removed by a helicase and a nuclease (not shown), allowing DNA polymerase to continue along the template to the 5' end of the next DNA fragment. (f) After completion of the new DNA strand, the nick between the two fragments is sealed by DNA ligase..

 



Role of initiators for initiation of DNA replication.
There are AT-rich and initiator-loading sequences on replicators. Initiators bind the initiator-loading sequences and at once the AT-rich sequences are rewound. Initiators recruit other proteins involved in DNA replication.



Preinitiation complex

In early S phase, S-Cdk and Cdc7 activation lead to the assembly of the preinitiation complex, a massive protein complex formed at the origin. Formation of the preinitiation complex displaces Cdc6 and Cdt1 from the origin replication complex, inactivating and disassembling the pre-replication complex. Loading the preinitiation complex onto the origin activates the Mcm helicase, causing unwinding of the DNA helix. The preinitiation complex also loads α-primase and other DNA polymerases onto the DNA.

After α-primase synthesizes the first primers, the primer-template junctions interact with the clamp loader, which loads the sliding clamp onto the DNA to begin DNA synthesis. The components of the preinitiation complex remain associated with replication forks as they move out from the origin.

 


Formation of pre-replication complex.
A schematic of the loading of the eukaryotic pre-replication complex.

 



Elongation

Elongation (W)

DNA polymerase has 5′–3′ activity. All known DNA replication systems require a free 3′ hydroxyl group before synthesis can be initiated (note: the DNA template is read in 3′ to 5′ direction whereas a new strand is synthesized in the 5′ to 3′ direction—this is often confused). Four distinct mechanisms for DNA synthesis are recognized:

  1. All cellular life forms and many DNA viruses, phages and plasmids use a primase to synthesize a short RNA primer with a free 3′ OH group which is subsequently elongated by a DNA polymerase.
  2. The retroelements (including retroviruses) employ a transfer RNA that primes DNA replication by providing a free 3′ OH that is used for elongation by the reverse transcriptase.
  3. In the adenoviruses and the φ29 family of bacteriophages, the 3′ OH group is provided by the side chain of an amino acid of the genome attached protein (the terminal protein) to which nucleotides are added by the DNA polymerase to form a new strand.
  4. In the single stranded DNA viruses—a group that includes the circoviruses, the geminiviruses, the parvoviruses and others—and also the many phages and plasmids that use the rolling circle replication (RCR) mechanism, the RCR endonuclease creates a nick in the genome strand (single stranded viruses) or one of the DNA strands (plasmids). The 5′ end of the nicked strand is transferred to a tyrosine residue on the nuclease and the free 3′ OH group is then used by the DNA polymerase to synthesize the new strand.


The first is the best known of these mechanisms and is used by the cellular organisms. In this mechanism, once the two strands are separated, primase adds RNA primers to the template strands. The leading strand receives one RNA primer while the lagging strand receives several. The leading strand is continuously extended from the primer by a DNA polymerase with high processivity, while the lagging strand is extended discontinuously from each primer forming Okazaki fragments. RNase removes the primer RNA fragments, and a low processivity DNA polymerase distinct from the replicative polymerase enters to fill the gaps. When this is complete, a single nick on the leading strand and several nicks on the lagging strand can be found. Ligase works to fill these nicks in, thus completing the newly replicated DNA molecule.

The primase used in this process differs significantly between bacteria and archaea/eukaryotes. Bacteria use a primase belonging to the DnaG protein superfamily which contains a catalytic domain of the TOPRIM fold type. The TOPRIM fold contains an α/β core with four conserved strands in a Rossmann-like topology. This structure is also found in the catalytic domains of topoisomerase Ia, topoisomerase II, the OLD-family nucleases and DNA repair proteins related to the RecR protein.

The primase used by archaea and eukaryotes, in contrast, contains a highly derived version of the RNA recognition motif (RRM). This primase is structurally similar to many viral RNA-dependent RNA polymerases, reverse transcriptases, cyclic nucleotide generating cyclases and DNA polymerases of the A/B/Y families that are involved in DNA replication and repair. In eukaryotic replication, the primase forms a complex with Pol α.

Multiple DNA polymerases take on different roles in the DNA replication process. In E. coli, DNA Pol III is the polymerase enzyme primarily responsible for DNA replication. It assembles into a replication complex at the replication fork that exhibits extremely high processivity, remaining intact for the entire replication cycle. In contrast, DNA Pol I is the enzyme responsible for replacing RNA primers with DNA. DNA Pol I has a 5′ to 3′ exonuclease activity in addition to its polymerase activity, and uses its exonuclease activity to degrade the RNA primers ahead of it as it extends the DNA strand behind it, in a process called nick translation. Pol I is much less processive than Pol III because its primary function in DNA replication is to create many short DNA regions rather than a few very long regions.

In eukaryotes, the low-processivity enzyme, Pol α, helps to initiate replication because it forms a complex with primase. In eukaryotes, leading strand synthesis is thought to be conducted by Pol ε; however, this view has recently been challenged, suggesting a role for Pol δ. Primer removal is completed Pol δ while repair of DNA during replication is completed by Pol ε.

As DNA synthesis continues, the original DNA strands continue to unwind on each side of the bubble, forming a replication fork with two prongs. In bacteria, which have a single origin of replication on their circular chromosome, this process creates a "theta structure" (resembling the Greek letter theta: θ). In contrast, eukaryotes have longer linear chromosomes and initiate replication at multiple origins within these.

 



Replication fork

Replication fork (W)



Scheme of the replication fork.
a: template, b: leading strand, c: lagging strand, d: replication fork, e: primer, f: Okazaki fragments
 
   

The replication fork is a structure that forms within the long helical DNA during DNA replication. It is created by helicases, which break the hydrogen bonds holding the two DNA strands together in the helix. The resulting structure has two branching "prongs", each one made up of a single strand of DNA. These two strands serve as the template for the leading and lagging strands, which will be created as DNA polymerase matches complementary nucleotides to the templates; the templates may be properly referred to as the leading strand template and the lagging strand template.

DNA is read by DNA polymerase in the 3′ to 5′ direction, meaning the nascent strand is synthesized in the 5' to 3' direction. Since the leading and lagging strand templates are oriented in opposite directions at the replication fork, a major issue is how to achieve synthesis of nascent (new) lagging strand DNA, whose direction of synthesis is opposite to the direction of the growing replication fork.

 


Many enzymes are involved in the DNA replication fork.

 



Leading strand

Leading strand (W)

The leading strand is the strand of nascent DNA which is synthesized in the same direction as the growing replication fork. This sort of DNA replication is continuous.

 



Lagging strand

Lagging strand (W)

The lagging strand is the strand of nascent DNA whose direction of synthesis is opposite to the direction of the growing replication fork. Because of its orientation, replication of the lagging strand is more complicated as compared to that of the leading strand. As a consequence, the DNA polymerase on this strand is seen to "lag behind" the other strand.

The lagging strand is synthesized in short, separated segments. On the lagging strand template, a primase "reads" the template DNA and initiates synthesis of a short complementary RNA primer. A DNA polymerase extends the primed segments, forming Okazaki fragments. The RNA primers are then removed and replaced with DNA, and the fragments of DNA are joined together by DNA ligase.

 



Dynamics at the replication fork

Dynamics at the replication fork (W)

In all cases the helicase is composed of six polypeptides that wrap around only one strand of the DNA being replicated. The two polymerases are bound to the helicase heximer. In eukaryotes the helicase wraps around the leading strand, and in prokaryotes it wraps around the lagging strand.



The assembled human DNA clamp, a trimer of the protein PCNA.
 
   

As helicase unwinds DNA at the replication fork, the DNA ahead is forced to rotate. This process results in a build-up of twists in the DNA ahead. This build-up forms a torsional resistance that would eventually halt the progress of the replication fork. Topoisomerases are enzymes that temporarily break the strands of DNA, relieving the tension caused by unwinding the two strands of the DNA helix; topoisomerases (including DNA gyrase) achieve this by adding negative supercoils to the DNA helix.

Bare single-stranded DNA tends to fold back on itself forming secondary structures; these structures can interfere with the movement of DNA polymerase. To prevent this, single-strand binding proteins bind to the DNA until a second strand is synthesized, preventing secondary structure formation.

Double-stranded DNA is coiled around histones that play an important role in regulating gene expression so the replicated DNA must be coiled around histones at the same places as the original DNA. To ensure this, histone chaperones disassemble the chromatin before it is replicated and replace the histones in the correct place. Some steps in this reassembly are somewhat speculative.

Clamp proteins form a sliding clamp around DNA, helping the DNA polymerase maintain contact with its template, thereby assisting with processivity. The inner face of the clamp enables DNA to be threaded through it. Once the polymerase reaches the end of the template or detects double-stranded DNA, the sliding clamp undergoes a conformational change that releases the DNA polymerase. Clamp-loading proteins are used to initially load the clamp, recognizing the junction between template and RNA primers.

 



DNA replication proteins

DNA replication proteins (W)

At the replication fork, many replication enzymes assemble on the DNA into a complex molecular machine called the replisome. The following is a list of major DNA replication enzymes that participate in the replisome:

 
Enzyme Function in DNA replication
DNA helicase Also known as helix destabilizing enzyme. Helicase separates the two strands of DNA at the Replication Fork behind the topoisomerase.
DNA polymerase The enzyme responsible for catalyzing the addition of nucleotide substrates to DNA in the 5′ to 3′ direction during DNA replication. Also performs proof-reading and error correction. There exist many different types of DNA Polymerase, each of which perform different functions in different types of cells.
DNA clamp A protein which prevents elongating DNA polymerases from dissociating from the DNA parent strand.
Single-strand DNA-binding protein Bind to ssDNA and prevent the DNA double helix from re-annealing after DNA helicase unwinds it, thus maintaining the strand separation, and facilitating the synthesis of the nascent strand.
Topoisomerase Relaxes the DNA from its super-coiled nature.
DNA gyrase Relieves strain of unwinding by DNA helicase; this is a specific type of topoisomerase
DNA ligase Re-anneals the semi-conservative strands and joins Okazaki Fragments of the lagging strand.
Primase Provides a starting point of RNA (or DNA) for DNA polymerase to begin synthesis of the new DNA strand.
Telomerase Lengthens telomeric DNA by adding repetitive nucleotide sequences to the ends of eukaryotic chromosomes. This allows germ cells and stem cells to avoid the Hayflick limit on cell division.

 



Replication machinery

Replication machinery (W)

Replication machineries consist of factors involved in DNA replication and appearing on template ssDNAs. Replication machineries include primosotors are replication enzymes; DNA polymerase, DNA helicases, DNA clamps and DNA topoisomerases, and replication proteins; e.g. single-stranded DNA binding proteins (SSB). In the replication machineries these components coordinate. In most of the bacteria, all of the factors involved in DNA replication are located on replication forks and the complexes stay on the forks during DNA replication. These replication machineries are called replisomes or DNA replicase systems. These terms are generic terms for proteins located on replication forks. In eukaryotic and some bacterial cells the replisomes are not formed.



E. coli Replisome. Notably, the DNA on lagging strand forms a loop. The exact structure of replisome is not well understood.
 
   

Since replication machineries do not move relatively to template DNAs such as factories, they are called a replication factory. In an alternative figure, DNA factories are similar to projectors and DNAs are like as cinematic films passing constantly into the projectors. In the replication factory model, after both DNA helicases for leading strands and lagging strands are loaded on the template DNAs, the helicases run along the DNAs into each other. The helicases remain associated for the remainder of replication process. Peter Meister et al. observed directly replication sites in budding yeast by monitoring green fluorescent protein (GFP)-tagged DNA polymerases α. They detected DNA replication of pairs of the tagged loci spaced apart symmetrically from a replication origin and found that the distance between the pairs decreased markedly by time. This finding suggests that the mechanism of DNA replication goes with DNA factories. That is, couples of replication factories are loaded on replication origins and the factories associated with each other. Also, template DNAs move into the factories, which bring extrusion of the template ssDNAs and nascent DNAs. Meister's finding is the first direct evidence of replication factory model. Subsequent research has shown that DNA helicases form dimers in many eukaryotic cells and bacterial replication machineries stay in single intranuclear location during DNA synthesis.

The replication factories perform disentanglement of sister chromatids. The disentanglement is essential for distributing the chromatids into daughter cells after DNA replication. Because sister chromatids after DNA replication hold each other by Cohesin rings, there is the only chance for the disentanglement in DNA replication. Fixing of replication machineries as replication factories can improve the success rate of DNA replication. If replication forks move freely in chromosomes, catenation of nuclei is aggravated and impedes mitotic segregation.

 



Termination

Termination (W)

Eukaryotes initiate DNA replication at multiple points in the chromosome, so replication forks meet and terminate at many points in the chromosome. Because eukaryotes have linear chromosomes, DNA replication is unable to reach the very end of the chromosomes. Due to this problem, DNA is lost in each replication cycle from the end of the chromosome. Telomeres are regions of repetitive DNA close to the ends and help prevent loss of genes due to this shortening. Shortening of the telomeres is a normal process in somatic cells. This shortens the telomeres of the daughter DNA chromosome. As a result, cells can only divide a certain number of times before the DNA loss prevents further division. (This is known as the Hayflick limit.) Within the germ cell line, which passes DNA to the next generation, telomerase extends the repetitive sequences of the telomere region to prevent degradation. Telomerase can become mistakenly active in somatic cells, sometimes leading to cancer formation. Increased telomerase activity is one of the hallmarks of cancer.

Termination requires that the progress of the DNA replication fork must stop or be blocked. Termination at a specific locus, when it occurs, involves the interaction between two components: (1) a termination site sequence in the DNA, and (2) a protein which binds to this sequence to physically stop DNA replication. In various bacterial species, this is named the DNA replication terminus site-binding protein, or Ter protein.

Because bacteria have circular chromosomes, termination of replication occurs when the two replication forks meet each other on the opposite end of the parental chromosome. E. coli regulates this process through the use of termination sequences that, when bound by the Tus protein, enable only one direction of replication fork to pass through. As a result, the replication forks are constrained to always meet within the termination region of the chromosome.

 



 
Regulation

Regulation

Regulation (W)

No text.

 



Eukaryotes

Eukaryotes (W)



The cell cycle of eukaryotic cells.
 
   

Within eukaryotes, DNA replication is controlled within the context of the cell cycle. As the cell grows and divides, it progresses through stages in the cell cycle; DNA replication takes place during the S phase (synthesis phase). The progress of the eukaryotic cell through the cycle is controlled by cell cycle checkpoints. Progression through checkpoints is controlled through complex interactions between various proteins, including cyclins and cyclin-dependent kinases. Unlike bacteria, eukaryotic DNA replicates in the confines of the nucleus.

The G1/S checkpoint (or restriction checkpoint) regulates whether eukaryotic cells enter the process of DNA replication and subsequent division. Cells that do not proceed through this checkpoint remain in the G0 stage and do not replicate their DNA.

After passing through the G1/S checkpoint, DNA must be replicated only once in each cell cycle. When the Mcm complex moves away from the origin, the pre-replication complex is dismantled. Because a new Mcm complex cannot be loaded at an origin until the pre-replication subunits are reactivated, one origin of replication can not be used twice in the same cell cycle.

Activation of S-Cdks in early S phase promotes the destruction or inhibition of individual pre-replication complex components, preventing immediate reassembly. S and M-Cdks continue to block pre-replication complex assembly even after S phase is complete, ensuring that assembly cannot occur again until all Cdk activity is reduced in late mitosis.

In budding yeast, inhibition of assembly is caused by Cdk-dependent phosphorylation of pre-replication complex components. At the onset of S phase, phosphorylation of Cdc6 by Cdk1 causes the binding of Cdc6 to the SCF ubiquitin protein ligase, which causes proteolytic destruction of Cdc6. Cdk-dependent phosphorylation of Mcm proteins promotes their export out of the nucleus along with Cdt1 during S phase, preventing the loading of new Mcm complexes at origins during a single cell cycle. Cdk phosphorylation of the origin replication complex also inhibits pre-replication complex assembly. The individual presence of any of these three mechanisms is sufficient to inhibit pre-replication complex assembly. However, mutations of all three proteins in the same cell does trigger reinitiation at many origins of replication within one cell cycle.

In animal cells, the protein geminin is a key inhibitor of pre-replication complex assembly. Geminin binds Cdt1, preventing its binding to the origin recognition complex. In G1, levels of geminin are kept low by the APC, which ubiquitinates geminin to target it for degradation. When geminin is destroyed, Cdt1 is released, allowing it to function in pre-replication complex assembly. At the end of G1, the APC is inactivated, allowing geminin to accumulate and bind Cdt1.

Replication of chloroplast and mitochondrial genomes occurs independently of the cell cycle, through the process of D-loop replication

 



Replication focus

Replication focus (W)

In vertebrate cells, replication sites concentrate into positions called replication foci. Replication sites can be detected by immunostaining daughter strands and replication enzymes and monitoring GFP-tagged replication factors. By these methods it is found that replication foci of varying size and positions appear in S phase of cell division and their number per nucleus is far smaller than the number of genomic replication forks.

P. Heun et al., (2001) tracked GFP-tagged replication foci in budding yeast cells and revealed that replication origins move constantly in G1 and S phase and the dynamics decreased significantly in S phase. Traditionally, replication sites were fixed on spatial structure of chromosomes by nuclear matrix or lamins. The Heun's results denied the traditional concepts, budding yeasts do not have lamins, and support that replication origins self-assemble and form replication foci.

By firing of replication origins, controlled spatially and temporally, the formation of replication foci is regulated. D. A. Jackson et al.(1998) revealed that neighboring origins fire simultaneously in mammalian cells. Spatial juxtaposition of replication sites brings clustering of replication forks. The clustering do rescue of stalled replication forks and favors normal progress of replication forks. Progress of replication forks is inhibited by many factors; collision with proteins or with complexes binding strongly on DNA, deficiency of dNTPs, nicks on template DNAs and so on. If replication forks stall and the remaining sequences from the stalled forks are not replicated, the daughter strands have nick obtained un-replicated sites. The un-replicated sites on one parent's strand hold the other strand together but not daughter strands. Therefore, the resulting sister chromatids cannot separate from each other and cannot divide into 2 daughter cells. When neighboring origins fire and a fork from one origin is stalled, fork from other origin access on an opposite direction of the stalled fork and duplicate the un-replicated sites. As other mechanism of the rescue there is application of dormant replication origins that excess origins do not fire in normal DNA replication.

 



Bacteria

Bacteria (W)



Dam methylates adenine of GATC sites after replication.
 
   

Most bacteria do not go through a well-defined cell cycle but instead continuously copy their DNA; during rapid growth, this can result in the concurrent occurrence of multiple rounds of replication. In E. coli, the best-characterized bacteria, DNA replication is regulated through several mechanisms, including: the hemimethylation and sequestering of the origin sequence, the ratio of adenosine triphosphate (ATP) to adenosine diphosphate (ADP), and the levels of protein DnaA. All these control the binding of initiator proteins to the origin sequences.

Because E. coli methylates GATC DNA sequences, DNA synthesis results in hemimethylated sequences. This hemimethylated DNA is recognized by the protein SeqA, which binds and sequesters the origin sequence; in addition, DnaA (required for initiation of replication) binds less well to hemimethylated DNA. As a result, newly replicated origins are prevented from immediately initiating another round of DNA replication.

ATP builds up when the cell is in a rich medium, triggering DNA replication once the cell has reached a specific size. ATP competes with ADP to bind to DnaA, and the DnaA-ATP complex is able to initiate replication. A certain number of DnaA proteins are also required for DNA replication — each time the origin is copied, the number of binding sites for DnaA doubles, requiring the synthesis of more DnaA to enable another initiation of replication.

In fast-growing bacteria, such as E. coli, chromosome replication takes more time than dividing the cell. The bacteria solve this by initiating a new round of replication before the previous one has been terminated. The new round of replication will form the chromosome of the cell that is born two generations after the dividing cell. This mechanism creates overlapping replication cycles.

 



 
Problems with DNA replication

Problems with DNA replication

Problems with DNA replication (W)

Main article: DNA replication stress

There are many events that contribute to replication stress, including:

 


Replication fork restarts by homologous recombination following replication stress.



Epigenetic consequences of nucleosome reassembly defects at stalled replication forks.

 



 
Polymerase chain reaction

Polymerase chain reaction

Polymerase chain reaction (W)

Researchers commonly replicate DNA in vitro using the polymerase chain reaction (PCR). PCR uses a pair of primers to span a target region in template DNA, and then polymerizes partner strands in each direction from these primers using a thermostable DNA polymerase. Repeating this process through multiple cycles amplifies the targeted DNA region. At the start of each cycle, the mixture of template and primers is heated, separating the newly synthesized molecule and template. Then, as the mixture cools, both of these become templates for annealing of new primers, and the polymerase extends from these. As a result, the number of copies of the target region doubles each round, increasing exponentially.

 



 
See also

 







 
  DNA replication (B)

DNA replication (B)

DNA replication (B)

DNA replication

DNA replication (B)

The Watson-Crick model of the structure of DNA suggested at least three different ways that DNA might self-replicate. The experiments of Matthew Meselson and Franklin Stahl on the bacterium Escherichia coli in 1958 suggested that DNA replicates semiconservatively. Meselson and Stahl grew bacterial cells in the presence of 15N, a heavy isotope of nitrogen, so that the DNA of the cells contained 15N. These cells were then transferred to a medium containing the normal isotope of nitrogen, 14N, and allowed to go through cell division. The researchers were able to demonstrate that, in the DNA molecules of the daughter cells, one strand contained only 15N, and the other strand contained 14N. This is precisely what is expected by the semiconservative mode of replication, in which the original DNA molecules should separate into two template strands containing 15N, and the newly aligned nucleotides should all contain 14N.

 


In semiconservative DNA replication an existing DNA molecule is separated into two template strands. New nucleotides align with and bind to the nucleotides of the existing strands, thus forming two DNA molecules that are identical to the original DNA molecule.
 

The hooking together of free nucleotides in the newly synthesized strand takes place one nucleotide at a time in the 5′ → 3′ direction. An incoming free nucleotide pairs with the complementary nucleotide on the template strand, and then the 5′ end of the free nucleotide is covalently joined to the 3′ end of a nucleotide already in place. The process is then repeated. The result is a nucleotide chain, referred to chemically as a nucleotide polymer or a polynucleotide. Of course the polymer is not a random polymer; its nucleotide sequence has been directed by the nucleotide sequence of the template strand. It is this templating process that enables hereditary information to be replicated accurately and passed down through the generations. In a very real way, human DNA has been replicated in a direct line of descent from the first vertebrates that evolved hundreds of millions of years ago.

DNA replication starts at a site on the DNA called the origin of replication. In higher organisms, replication begins at multiple origins of replication and moves along the DNA in both directions outward from each origin, creating two replication “forks.” The events at both replication forks are identical. In order for DNA to replicate, however, the two strands of the double helix first must be unwound from each other. A class of enzymes called DNA topoisomerases removes helical twists by cutting a DNA strand and then resealing the cut. Enzymes called helicases then separate the two strands of the double helix, exposing two template surfaces for the alignment of free nucleotides. Beginning at the origin of replication, a complex enzyme called DNA polymerase moves along the DNA molecule, pairing nucleotides on each template strand with free complementary nucleotides. Because of the antiparallel nature of the DNA strands, new strand synthesis is different on each template. On the 3′ → 5′ template strand, polymerization proceeds in the 5′ → 3′ direction, and this growing strand is called the leading strand. However, polymerization must be carried out differently on the 5′ → 3′ template strand because nucleotides cannot be assembled in the 3′ → 5′ direction. Here short sequences of RNA are polymerized on the template. These sequences act as primers to which the DNA polymerase can add nucleotides in the 5′ → 3′ direction but in the opposite direction in which synthesis is proceeding on the lagging strand. The DNA polymerase hence makes short segments of DNA called Okazaki fragments in the “wrong” direction. For this reason the strand synthesized on the 5′ → 3′ template strand is called the lagging strand. Later, the RNA primers are removed and the Okazaki fragments are joined. This RNA priming system cannot be used to synthesize the very end of the 3′ → 5′ strand; once the last RNA primer is removed, synthesis cannot continue over the remaining gap. To overcome this obstacle, the enzyme telomerase adds multiple copies of a nucleotide sequence to the end of the DNA strand to allow completion of replication. Despite the peculiar events on the lagging strand, the entire DNA strand is eventually polymerized, and the two daughter DNA molecules thus produced are identical.

 


DNA replication in higher organisms begins at multiple origins of replication and progresses in two directions..

 



 







 
 

 


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