PTT 207 Biomolecular and Genetic Engineering Semester 2 2013/2014 BY: PUAN NURUL AIN HARMIZA ABDULLAH
INTRODUCTION
INTRODUCTION DNA replication involves: • The melting apart of the two strands of the double helix followed by the polymerization of new complementary strands. • Decisions of when, where, and how to initiate replication to ensure that only one complete and accurate copy of the genome is made before a cell divides.
STEPS IN PROKARYOTES DNA REPLICATION
Step 1: Initiation: Unwinding of the double helix: • DNA Replication begins at the Origin of Replication (usually A-T rich). • Helicase cuts hydrogen bonds and separates DNA in half. • Single Strand Binding Proteins (ssB) attach to the halfs and keep the DNA molecules separated (they are needed because the sides are attracted to each other and without the ssB they would move back together). • The Replication Fork is forms with the Leading and Lagging strands.
Step 2: Elongation: Assembly of 2 new strands of DNA: • DNA polymerase (which is the enzyme that inserts itself into the space between the two strands) attaches new nucleotides to the free 3’ hydroxyl end. This imposes two conditions on the elongation process: 1. First, replication can only take place in the 5’ and 3’ direction ( leading strand ). 2. Second, a short strand or RNA known as a primer must be available to serve as the starting point for the attachment of new nucleotides. The primer simply gets the bases primed to receive new bases that will form the new DNA strand.
Step 2: Elongation: Assembly of 2 new strands of DNA: • During replication, much of the newly formed DNA is found in short fragments of one to two thousand nucleotides in prokaryotes and a few hundred nucleotides in eukaryotes. • These fragments are known as Okazaki Fragments . • These fragments occur during the elongation of the daughter DNA strand that must be built in the 3’ to 5’ direction ( lagging strand ).
Step 2: Elongation: Assembly of 2 new strands of DNA: • There are 2 strands during the replication process. They are: • Leading Strand: • Continuous elongation process in the 5’ and 3’ direction. • Same direction as the movement of the replication fork. • Lagging Strand: • More slowly than the leading strand. • DNA polymerase adds Okazaki Fragments which are eventually spliced together by the enzyme DNA ligase. • Opposite direction to the movement of the replication fork.
Step 2: Elongation: Assembly of 2 new strands of DNA: • Another enzyme called primase synthesizes an RNA primer to begin the elongation process. Only one primer is needed on the leading strand. A new primer is needed for each Okazaki Fragment on the lagging strand.
DNA polymerases • Can only add nucleotides in the 5′→3′ direction. Why don't DNA polymerases elongate chains in the 3' to 5' direction? • Cannot initiate DNA synthesis de novo. • The key feature of DNA polymerases is that they cannot initiate DNA synthesis; but require a “ primer ” to get started. • Require a primer with a free 3′ -OH group at the end.
Problem • DNA polymerases can only add nucleotides from 5′→3′ but, the two strands of the double helix are antiparallel. Solution • Semidiscontinuous replication.
Semidiscontinuous DNA replication • Major form of replication in eukaryotic nuclear DNA, some viruses, and bacteria.
• Discontinuous replication occurs on the 5′→3′ template strand (lagging strand). • DNA is copied in short segments called “Okazaki fragments” moving in the opposite direction to the replication fork. • Repetition of primer synthesis and formation of Okazaki fragments. • Lagging strand replication requires the repetition of 4 steps: primer synthesis elongation primer removal with gap filling joining of Okazaki fragments
Synthesis of both strands occurs concurrently • Nucleotides are added to the leading and lagging strands at the same time and rate. • Two DNA polymerases, one for each strand.
Step 3: Termination: • Once the new strands are complete, the daughter DNA molecules rewind automatically back to their original helix structure. • The problem with the end of a linear chromosome with the RNA primer has been removed from the 5’ end of each daughter strand, there is no adjacent fragment onto which new DNA nucleotides can be added to fill the gap . The result is that each replication results in slightly shorter daughter chromosomes. • Eukaryotes have special buffer zones called telomeres at the end of each chromosome to guard against this problem. These are highly repetitive nucleotide sequences typically rich in G nucleotides . These regions do not direct cell development.
Step 4: Proofreading and Correction: • This step of DNA replication ensures accuracy of replication . • After each new nucleotide is added to a new DNA strand, DNA polymerase can recognize whether or not hydrogen bonding is taking place between base pairs. • Absence of hydrogen bonding indicates a mismatch . DNA polymerase excises the incorrect base and then adds the correct nucleotide.
MULTI-PROTEIN MACHINES MEDIATE BACTERIAL DNA REPLICATION
Bacterial DNA polymerases have multiple functions DNA polymerase I • Primer removal, gap filling between Okazaki fragments, and nucleotide excision repair pathway. • Two subunits: Klenow fragment has 5 ′→ 3 ′ polymerase activity; other subunit has both 3 ′→ 5 ′ and 5 ′→ 3 ′ exonuclease activity (which can remove the dNMP from the end of the DNA chain by breaking the terminal phosphodiester bond = used in “proofreading”) . • Proofreading = If the polymerase make mistake, it can backtrack to remove the nucleotide with a mismatched base. • Unique ability to start replication at a nick in the DNA sugar- phosphate backbone. • Used extensively in molecular biology research.
DNA polymerase III • Main replicative polymerase. DNA polymerase II • Involved in DNA repair mechanisms. DNA polymerases IV and V • Mediate translesion synthesis (Chapter 7).
Is leading strand synthesis really continuous? • DNA polymerase III can be blocked by a damaged site on the template DNA. • Sometimes DNA polymerase collides with RNA polymerase and is stalled. • In both cases, replication can be jumpstarted on the leading strand by formation of a new primer at the replication fork.
Please watch the DNA replication mechanism at these urls: 1. http://highered.mcgraw- hill.com/sites/9834092339/student_vie w0/chapter14/dna_replication.html 2. http://www.wiley.com/college/pratt/04 71393878/student/animations/dna_rep lication/index.html 3. http://www.youtube.com/watch?v=teV 62zrm2P0
http://www.youtube.com/watch?v=EYGrElVyHnU
SUPERCOILING
Relaxed State is No Bend
L, T, and W characterize superhelical DNA • L= linking number = number of times one strand wraps around the other. It is an integer for a closed circular DNA. • T = twists/turns in the DNA T = 26 (No. bp/10.4; positive for W = 0 right-handed DNA) • W = writhes =number of turns of the helix around the L = T + W superhelical axis
Linking Number
Separation of DNA strands = Supercoiling in advance of separation
Positively supercoiling (left handed): T = 0, W = 0, then L = 0 T = +3, W = 0, then L = +3 T = +2, W = +1, then L = +3 Negatively supercoiling (right handed): T = 0, W = 0, then L = 0 T = -3, W = 0, then L = -3 T = -2, W = -1, then L = -3 Negative supercoils favor local unwinding of the DNA, allowing processes such as transcription, DNA replication, and recombination
If the protein has topoisomerase activity, it will relax most of the supercoiled plasmid DNA. Relaxed circular DNA is less compact and runs more slowly in a gel than supercoiled circular DNA, and thus will remain closer to the negative electrode.
Movement of the replication fork machinery results in: • Positive supercoiling ahead of the fork. • Negative supercoiling in the wake of the fork. • Torsional strain (resistance to bond twisting) that could inhibit fork movement is relieved by DNA topoisomerase.
Topoisomerases are required to relieve torsional strain • Topoisomerases I : • breaks only one strand • relaxes negatively supercoiled DNA • introduces a change of increments of 1 in writhe • Topoisomerase II : • breaks both strands • relaxes both negative and positively supercoiled DNA • introduces a change in increments of 2 in writhe
Topoisomerases I
Topoisomerase II
Replication of the circular genome of bacteria is a highly coordinated, dynamic process, requiring many specialized enzymes and other proteins. How does it compare with replication of much larger, linear eukaryotic genomes?
EUKARYOTIC DNA REPLICATION
• The replication machinery in eukaryotic DNA replication is a much larger complex, coordinating many proteins at the site of replication, forming the replisome . • The replisome is responsible for copying the entirety of genomic DNA in each proliferative cell.
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