PTT 207 Biomolecular and Genetic Engineering Semester 2 2013/2014 - - PowerPoint PPT Presentation

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.

• 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.

Why don't DNA polymerases elongate chains in the 3' to 5' direction?

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
• f 4 steps:

primer synthesis  elongation  primer removal with gap filling  joining of Okazaki fragments

Synthesis of both strands

• ccurs 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
• f 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
• f times one strand wraps

around the other. It is an integer for a closed circular DNA.

• T = twists/turns in the DNA

(No. bp/10.4; positive for right-handed DNA)

• W = writhes =number of

turns of the helix around the superhelical axis

T = 26 W = 0 L = T + W

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.

Eukaryotic origins of replication

• Multi-protein machines trade places

during eukaryotic DNA replication.

• Mice have 25,000 origins, spanning ~150

kb each.

• Humans have 10,000 to 100,000 origins.

• The overall rate of replication is largely

determined by the number of origins used and the rate at which they initiate.

• Replication forks are clustered in

“replication factories.”

• Forty to many hundreds of forks are active

in each factory.

• Histones removal at origin of replication
• Pre-Replication Complex Formation
• Assembly of the origin recognition complex (ORC)
• Assembly of the replication licensing complex
• RNA priming of leading strand and lagging strand DNA synthesis
• Polymerase switching
• Elongation of leading strands and lagging strands
• PCNA: a sliding clamp with many proteins partners
• Proofreading
• Histone deposition
• Topoisomerase untangles the newly synthesis DNA

involves the ordered assembly of additional replication factors to unwind the DNA and accumulate the multiple eukaryotic DNA polymerases around the unwound DNA

PREREPLICATION COMPLEX FORMATION

DUPLEX UNWINDING AT REPLICATION FORKS AND RNA PRIMING OF LEADING AND LAGGING STRAND SYNTHESIS

POLYMERASE SWITCHING AND STRAND ELONGATION

FILL-IN GAPS LEFT BY PRIMER REMOVAL AND JOINING OF OKAZAKI FRAGMENTS

DIFFERENCES BETWEEN EUKARYOTIC AND PROKARYOTIC DNA REPLICATION

PROKARYOTE EUKARYOTE Occurs inside cytoplasm Occurs inside nucleus Only 1 origin of replication per DNA molecule Origin of replication are many (over 1000) in each chromosome Origin of replication is formed of about > 100-200 nucleotides Each origin of replication is formed of about 150 nucleotides Replication of DNA occurs at 1 point in each prokaryotic chromosome Replication occurs at several points simultaneously in each chromosome Only 1 replication fork is formed in each replicating prokaryotic chromosome A number of replication forks are formed simulataneously in each replication DNA Chromosome has 1 replicon Have large numbers of replicons (>50K), but replication does not

• ccur simultaneously on all

replicons

PROKARYOTE EUKARYOTE No replication bubble is formed during replication A number of replication bubbles are formed in 1 replication DNA molecule Initiation of DNA replication is carried out by protein DnaA and DnaB Initiation of DNA replication is carried out by multisubunit protein, origin recognition complex DNA gyrase is needed DNA gyrase is needed Okazaki fragments are large, 1000-2000 nucleotides long Okazaki fragments are short, 100-200 nucleotides long Replication is very rapid, 2000bp per second are added Replication is slow, 100 nucleotides per second are added

6.7 Telomere maintenance: the role of telomerase in DNA replication, aging, and cancer

Will be presented by Amirah

The End

Thank You