Chapter 17: DNA Synthesis: Doubling Your Genetic Stuff summary

What is DNA replication?

• It is the process by which cells copy their DNA.
• It occurs in a cell going to reproduce itself by either meiosis or mitosis.
• Mutations, mistakes in the DNA sequence, can affect the cell’s normal function.
• The cells have a process of the parent DNA code and copying the DNA into a new DNA strand.

What Enzymes are involved in DNA replication?

• Helicase enzyme breaks the hydrogen bonds that hold the double helix DNA together.
• Topoisomerase enzyme breaks and facilitates sealing of the DNA to remove tension resulting from twisting.
• Primase enzyme helps in the production of primers.
• DNA polymerase manufactures a new DNA  strand. There are two DNA polymerases:
  • DNA polymerase III copies DNA from the parent strand to build a new strand that is complementary to the parent strand.
  • DNA polymerase I enzyme replaces RNA nucleotides from RNA primers with DNA nucleotides.
• DNA ligase enzyme joins the gaps in the DNA backbone by generating covalent bonds between the DNA nucleotides.

Replication fork (Initiation)

• An enzyme complex is the group of all enzymes involved in the replication process.
• DNA replication starts at defined regions within chromosomes.
• These regions on found om DNA sequences are called origins of replication.
• Specific proteins can identify this sequence and open up the double-stranded DNA at the origin of replication.
• Eukaryotic chromosomes have several origins of replication.
• A replication bubble is a visible loop that forms in the double helix DNA  after being unwound by an enzyme.
• A replication fork is a V-shaped structure that forms when DNA double helix is unwound on both sides of the replication bubble.

How helicase enzyme unwinds DNA double helix strand

• After DNA has been unwound at the origin f replication, helicase enzyme promotes breaking of hydrogen bonds that hold DNA strands together.
• Helicase digests the hydrogen bonds and the two DNA strands separate.
• Single-stranded binding proteins then bind on the single strands to prevent them from rejoining together.
• Topoisomerase enzymes remove positive supercoils cutting the strand to relax it and rejoining it again.

Elongation

• Primase enzymes make short RNA strands called primers that are used in the generation of a new DNA strand.
• DNA polymerase III enzymes start making new DNA strands by attaching DNA nucleotides to the primer’s  3’ end.
• The new strand grows from 5’ to 3’ direction,
• DNA polymerase III processivity has a β subunit that encircles the DNA strand and moves along the DNA template.
• DNA polymerase I move along the DNA strand, removes RNA nucleotides and replaces them with DNA nucleotides.
• DNA synthesis uses dNTPs to generate a new DNA strand.
• DNA polymerase III moves from one primer to another primer without forming covalent bonds. This leaves gaps between the last nucleotide and the 5’ end of the new strand.
• DNA ligase moves along the new DNA strand and seals the gaps by forming covalent bonds in the sugar-phosphate backbone.

Termination

• Enzyme complexes in the DNA replication process catalyze the replication of DNA in both directions of the replication fork.
• When the replication forks meet, the enzyme complex is released from the DNA.
• Template sequence misreading can cause mutations.
• Apart from  5’ to 3’ polymerase activity, DNA polymerase has a 3’ to 5’ exonuclease activity.

Leading and lagging strands of DNA

• The leading strand is the new DNA sequence being synthesized from the 5’ to 3’ direction by DNA polymerase.
• The DNA polymerase also copies DNA in the opposite direction (3’ to 5’ direction).
• Because of the 5’ to 3’ activity of the DNA polymerase, the opposite strand is synthesized in short sequences called Okazaki fragments.
• DNA polymerase I remove primers on the lagging strand.
• DNA ligase forms covalent bonds within the DNA fragment on the lagging strand.

Revision

Requirements for DNA Replication
Substrates ( Precursors )
Template
Primer
Enzymes

The four substrates are
The four deoxynucleoside triphosphate, dATP, dGTP, dCTP, dTTP

From where do we get the energy necessary for the addition of nucleoside monophosphate
Cleavage of the bond between the alpha and the beta phosphate of the substrate which produces a pyrophosphate bond

Semi-Conservative DNA replication
Each new DNA molecule is composed of a one parenteral stand and one daughter strand

Primers
DNA Polymerase cannot start without it – Oligonucleotides 10/60 bases – RNA in nature – Added to the 3′ end of the DNA by Primase – Provides a free 3′ -OH

Enzymes are required for
Initiation
Elongation
Termination
Proof Reading

Replicons
Numerous sites in which replication starts – goes in both directions until neighboring replicons fuse

Direction of replication :
5′ to 3′ direction – new nucleotide can be attached only to the free 3′ OH end

Steps involved in DNA replication

1. Identification of the origin of replication 2. Unwinding 3. Replication fork formation 4. Initiation and elongation 5. formation of replication bubbles + ligation of newly synthesized DNA segments 6. reconstitution of chromatin structure

Identification of the origin of replication
Association between : dsDNA binding protein (DBP) + 150-250 bp = complex → local denaturation + unwinding of an adjacent A-T rich region

Whats the function of Single-Stranded Binding Protein ( SSBB )
Stabilizes the complex and prevents recoiling ( unwinding )

Formation of Replication Fork

1. Helicase unwinding short segment 2. SSBB preventing recoiling 3. Primase synthesizes Primer 4. DNA polymerase III initiates strand synthesis

How DNA replication is happening on the lagging strand
The DNA is synthesized in short fragments called Okazaki fragments

Okazaki fragments
newly synthesized DNA fragments attached to an RNA initiator

What happens after many Okazaki fragments are generated

1. Removal of RNA primers 2. Filling the gaps with proper base-paired deoxynucleotide 3. Ligation of the fragments by DNA ligase

In replication of mitochondrial genome what happens to RNA primers
Remains as integral part of the circular DNA structure

DNA gyrase role in replication
Remove DNA superteists

DNA helicase role in replication
Untwisting of DNA double helix

DBP role in replication
Stabilize unwinding

Primase role in replication
Synthesize RNA primers

DNA polymerase III role in replication
Synthesize DNA

DNA polymerase I role in replication
Erase RNA primers & Fill gaps

DNA ligase role in replication
Joins the ends of DNA

Chromatin consists of
Very long dsDNA + Histones + Non histones proteins ( some acidic and larger ) + Small quantity of RNA

Histones are
Positively charged proteins → make ionic bonds with negative phosphate groups in the nucleic acid – H1, H2A, H2B, H3, H4

Three levels of DNA coiling
Nucleosomes : DNA wraps around nucleosome core particles
2nd level : H1 binds to DNA + other Histones
Loop formation : DNA forms loops radiating from the central scaffold of non-histone proteins

Nucleosome core particles
146 BP of DNA wrapped around the histone octamer ( 2 copies each of the core histones H2A, H2B, H3, H4 )

Function of DNA topoisomerase 1
Helps in supercoiling

what does it mean that replication is semiconservative?
because both parental strands are still intact but are no longer together

how does DNA synthesis in prokaryotes occur
starts with the binding of the protein DnaA at a single point of origin, OriC

the two parental strands are copied simultaneously with protein help (gyracase, helicase, and and single strande binding proteins)

synthesis begins at two asymmetrical Y-shaped replication forks and moves bidirectionally

how is replication initiated in in prokaryotes
dnaA and the primosome dnaB (dna Helicase) and dnaG (dna primase)

what proteins function in unwinding of the parental strands
helicases (e.g. DnaB)–unwinds DNA

Single stranded binding protein–prevents strands form reassociating and protects Dna from single strand enzymatic cleavage

Topoisomerase–break phosphodiester bonds and relieve supercoiling (DNA gyrase)

How is DNA synthesized at the replication fork in prokaryots
DNA polymerase moves 3′ to 5′ and synthesis the DNA 5′ to 3′ prime.

incoming nucleotides form a base pair with its complementary nucleotide on the template strand, then an ester bond is formed between the first 5′ phosphate of the incoming nucleotide and the free 3′-hydroxyl (OH) group at the end of the growing chain; pyrophosphate is released providing energy to drive the polymerization process

leading strand is continous and lagging strand short okazaki fragements are created by DNA pol 1. off DNA primase RNA primers and then removes the RNA primers. ligase connects DNA strands

what enzyme synthesises the short RNA primers made on the lagging strain. also can join two nucleoside triphosphates.
DNA primase

what is the function of DNA ligase
joining okazaki fragements

What fixes base pair errors
Pol 3 has proofreading function

the three steps of high fidelity DNA synthesis

how many pol. does E. Coli have and what are there functions

what is processivity
DNA pol. stays attached to parental chain as it moves down chain

difference between eukaryotic and prokaryotic replication
nculeosomes dissasemble .different regions on the same chromosome replicate at distinct times in S phase of the cell cycle (unlike prokaryotes that can replicate their DNA continuously) ; highly condensed chromatin (heterochromatin) replicates late, while less condensed chromatin (euchromatin) replicates early. new nucleosomes are assembled behind the replication fork