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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