DNA Replication - Tusculum University
DNA REPLICATION
SEMICONSERVATIVE REPLICATION
1 Conservative replication
1 This model, which was shown to be incorrect, predicted that after replication, the parent double-stranded DNA would remain intact while the daughter double-stranded DNA would be entirely newly synthesized
2 Semiconservative replication
1 This model predicts that both daughter DNA molecules would contain one strand each from the parent DNA and one entirely newly synthesized strand
Enzymology of DNA Replication
1 DNA polymerases
1 Substrates
1 Needs all four deoxynucleoside 5'-triphosphates
1 5' mono- and di- phosphates and 3' mono-, di-, or tri-phosphates do not work
2 Needs a DNA template
3 Needs a nucleotide primer with free 3'-OH group
1 All nucleic acids are synthesized in the 5' to 3' direction
2 Reaction
1 Poly(deoxynucleotide)n-3'-OH + dNTP ( Poly(deoxynucleotide)n+1-3'-OH + PPi
2 Monophosphates will not work
1 Formation of the phosphodiester bond is extremely endergonic
2 Energy released from breaking pyrophosphate from NTP provides the energy for polymerization
3 Prokaryotic DNA polymerases
1 DNA polymerase I
1 3' to 5' polymerase activity
1 Catalyze DNA polymerization
2 5' to 3' exonuclease activity
1 Nucleotides are removed from 5'-P terminus
2 Functions to remove ribonucleotide primers
3 Functions to remove RNA primer and replace it with deoxyribonucleotides
2 DNA polymerase II
1 Used in DNA repair
2 Has 5' - 3' polymerase activity and 3' - 5' exonuclease activity
3 DNA polymerase III
1 Major enzyme used in replication
2 Has 5' - 3' polymerase activity and 3' - 5' exonuclease activity
4 Proofreading
1 If an incorrect base is added to the growing DNA chain DNA polymerases can back up and remove that base and then continue
1 The removal of the incorrect base is referred to as 3' to 5' exonuclease activity
2 DNA ligase
1 Function
1 Forms phosphodiester bonds between two segments of DNA
2 Mechanism
1 Joins 3'-OH to a 5'-monophosphate group
3 DNA Gyrase
1 Function
1 Unwinds DNA helix into single-stranded DNA so that replication can proceed
4 Primase / RNA polymerase
1 Function
1 DNA polymerase must connect nucleotides to 3'-OH group
1 Cannot lay down first nucleotide
2 RNA polymerase does not have this requirement
1 A few ribonucleotides laid down by RNA polymerase can serve as a primer for DNA polymerase
2 RNA polymerase
1 Primes the leading (continuous strand)
3 Primase
1 Primes lagging (discontinuous strand)
4 Primer
1 1 to 60 bases
2 Provides 3'-OH group for DNA polymerase III to add a deoxynucleotide
TOPOGRAPHY OF DNA REPLICATION
1 Unwinding of parental DNA during replication causes stress in the unreplicated portion of DNA, which if not relieved, could prevent the replication fork from moving upstream
1 Bacterial chromosomes cannot relieve their stress as it is a covalently closed circle
2 Eukaryotic chromosomes, though linear, are too large to rotate to relieve stress
2 DNA topoisomerases
1 DNA gyrase, a topoisomerase, uses breaking, twisting, and ligating ability to remove stress
1 DNA gyrase wraps DNA around it, cuts both strands of DNA. then passes DNA through the gap of broken strands and reforms the phosphodiester backbone
replication fork
1 Origin of replication
1 DNA replication begins at specific regions of DNA referred to as 'Origins of Replication' or ori sites
1 Prokaryotes contain only one ori site
2 Eukaryotes contain multiple ori sites per chromosome
1 Multiple ori sites are needed due to the larger size of DNA in eukaryotes and the slower speed of DNA replication of eukaryotic DNA polymerases
2 Replication forks
1 DNA is replicated bi-directionally from each ori site
2 A replication fork is the area of DNA that is being unwound prior to replication
3 There are two replication forks for every one ori
1 As DNA replication begins continuously on one strand, the first Okazaki fragment produced becomes the leading strand for the other replication fork
3 Advance of the replication fork and unwinding the helix
1 Addition to nucleotides and unwinding of DNA are two different processes
1 DNA polymerase III cannot unwind DNA
2 Unwinding is catalyzed by enzymes called helicases
2 Single-stranded binding proteins
1 DNA polymerase III is not directly behind the helicase
1 There is therefore some single-stranded DNA in the leading strand
2 There is a larger gap of single stranded-DNA on the lagging strand
2 Single-stranded binding proteins coats single-stranded DNA so they cannot reform hydrogen bonds
1 These single-stranded binding proteins must be displaced by DNA polymerase III or another enzyme
CONTINUOUS REPLICATION
1 DNA helicase
1 Unwinds DNA double helix
1 Separates double-stranded DNA into single-stranded sections
2 Starts at ori site
3 Results in topographical stress
2 Single-stranded DNA binding proteins
1 Keeps complimentary strands of DNA from reannealing
3 DNA topoisomerases (e.g., DNA gyrase)
1 Relieves stress caused by helicases
4 Primase (RNA polymerase)
1 Lays down RNA primer
5 DNA Polymerase III
1 Adds nucleotides to 3’ end of primer
2 Adds nucleotides to 3’ end of growing DNA polymer
6 DNA Ligase
1 Seals the ends of the newly created DNA circle
Discontinuous replicatioN
1 Replication fork
1 At the replication fork, one strand is synthesized continuously, the other discontinuously, because the strands are antiparallel
1 All DNA is synthesized in the 5' to 3' direction
2 Steps
1 DNA is unwound
2 RNA primer is made at fork
3 DNA polymerase adds nucleotides to 3’ end
4 DNA polymerase runs into previous primer
5 Cycle starts over again (1 through 4)
6 DNA polymerase I removes RNA primer and replaces it with deoxynucleotides
3 Okazaki fragments
1 Size
1 Eukaryotes
1 100 - 200 bases
2 Prokaryotes
1 1000 - 2000 bases
2 Connecting Okazaki fragments
1 Okazaki fragments are joined to form a continuous DNA containing no ribonucleotide
2 DNA polymerase I
1 Removes the primer ribonucleotides
2 Replaces them with deoxyribonucleotides
3 DNA ligase
1 Catalyzes formation of phosphoester bond between nucleotides
4 No 3' to 5' polymerase
1 It would be simpler, however, evolution has not taken this course
Eukaryotes
1 Rate
1 Eukaryotic DNA polymerases are slower than bacterial DNA polymerases
1 Can replicate about 500 - 5,000 bases per minute
2 Bacterial can replicate about 105 (100,000) per minute
2 To make up for the slower replication, eukaryotes have more origins of replication
1 Mammals have around 12,000 ori sites
2 DNA polymerases
1 DNA polymerase (
1 Polymerizes the discontinuous strand
2 DNA polymerase (
1 Is used in DNA repair
3 DNA polymerase (
1 Polymerizes the continuous strand
4 DNA polymerase (
1 Found in mitochondria and chloroplasts
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