Chemistry of DNA synthesis

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Chemistry of DNA synthesis

Substrates for DNA synthesis:

•      Deoxyribonucleotide triphosphates (dNTPs)

•      Primer: template junction

The primer provides a free 3’-OH while template provides a single-stranded DNA to be copied.

DNA polymerization is a NUCLEOPHILIC SUBSTITUTION (S_N2) REACTION.

DNA polymerase has 3 main domains:

–     Palm domain

–     Thumb domain

–     Finger domain

v  Palm domain:

•      Possess the active site of DNA synthesis and is composed of a β-sheet.

•      The active site in the palm domain can distinguish between rNTPs and dNTPs.

•      The rNTPs are present in around 10 fold higher concentrations in the cell.

•      But the nucleotide-binding pocket is too small for the 2’-OH on the incoming rNTP.

•      Thus the polymerase can exclude the rNTPs by steric constraining.

•      Correct base pairing is also required for catalysis.

•      If an incorrect dNTP comes, its α-phosphoryl group cannot properly align with the 3’-OH of the growing strand.

•      Once the correct dNTP is bound in the pocket, the reaction can continue.

•      The palm domain also binds Zn²⁺ and Mg²⁺ which are crucial for catalysis.

v  Finger domain:

•      Composed of α-helix.

•      Once the correct dNTP is bound in the pocket, the finger domain moves to enclose the base-paired dNTPs.

•      This conformational change brings the dNTP and the primer (or growing DNA strand) into correct orientation with the divalent metal ions.

•      The O-helix of the finger domain moves 40° to enclose the base by stacking interaction with its tyrosine residue.

•      Metal ion A helps to deprotonate the 3’-OH of the primer producing an oxyanion.

•      This oxyanion attacks the α–phosphoryl group of the incoming dNTP.

•   Metal ion B coordinates the negative charge of the β- and γ-phosphate groups and stabilizes the pyrophosphate leaving group.

•      Lysine and arginine residues on the finger domain also help to stabilize the pyrophosphate and the tyrosine residue holds the dNTP in place for catalysis (stacking interaction).

•   The finger domain also associates with the template region resulting in a 90° turn in the template which helps to avoid confusion in the active site.

•      This ensures that only one template nucleotide remains in the active site.

v  Thumb domain:

•      It is not intimately involved in catalysis.

•   It interacts with the DNA that has been synthesized most recently and holds the primer: template junction in the active site.

•      This reduces the dissociation of the polymerase from the template.

Proofreading

•     If an incorrect nucleotide is incorporated by DNA polymerase (frequency of error is 10^-6), it is recognized immediately.

•      The 3’ → 5’ exonuclease activity of the polymerase then excise the incorrect nucleotide from the new strand.

•      The polymerase then resumes its forward motion and inserts the correct nucleotide.

•      The palm domain also has proofreading activity.

•      It H-bonds with the base pairs in the minor groove. It is not sequence-specific but occurs only when the nucleotides are correctly base-paired.

•      If a mismatch occurs, replication slows down and the palm domain is not able to make contact with the minor groove.

•      This frees the primer: template junction to move and make contact with the exonuclease site.

•      The exonuclease site removes the incorrect base from 3’ to 5’ direction in a process called proofreading.

•      After excision is complete, the primer: template junction slides back to the replication active site.

Watch animations on DNA Replication Overview from Pearson

Watch animations on Chemistry of DNA Synthesis from Pearson

Cloning Vectors

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Cloning Vectors

A self-replicating DNA molecule attached with a foreign DNA fragment to be introduced into a cell.

Has features that make it easier to insert DNA and select  for the presence of vector in the  cell:

—Origin of replication

—Antibiotic resistance gene

—Cloning site (MCS)

—Promoters and terminators for expression of the cloned  gene

Types of cloning vectors

1.Plasmids (10-15kb inserts)

2.Bacteriophage (bacterial viruses), 30-50kb inserts

3.Cosmids (35-50kb insert)

4.Bacterial Artificial Chromosomes (BACs)

o Use fertility F plasmid

o 75-300kb inserts possible

o Developed during the human genome project

5.Yeast Artificial Chromosomes (YACs)

o Mimics yeast chromosome

o Contains all regions for replication (yeast ori and centromere)

o 100-1000kb inserts

o Developed during the human genome project

Plasmid

# Plasmids  are  double-stranded,  closed,  circular  DNA  molecules which exist in the host cell as extrachromosomal units.

# They are self-replicating, can be single or multi-copy per cell.

# Some plasmids are under relaxed replication control thus permitting accumulation of huge copy numbers (up to 1000 copies per cell). These are preferred in cloning because of their high yield.

# 1-200 kb in size

# Depend on the host proteins for replication and maintenance.

# All naturally occurring plasmids do not always contain all the  essential properties of a suitable cloning vector.

Plasmid Cloning Vectors

# Derived from naturally occurring plasmids

# Altered features:

# smaller size (removal of non-essential DNA) 

# a higher transformation efficiency, easier manipulation and purification

# unique restriction enzyme sites

# one or more selectable markers

# other features:  promoters, terminators, etc.

# Can hold up to 10 kb fragments

pBR322

# One of the most widely used standard artificial cloning vector

#It is a 4.36kb double-stranded plasmid vector originating from fragments of three naturally occurring plasmids

# It has genes for resistance against two antibiotics: tetracycline and  ampicillin

# It contains 20 RE sites, six of which are located within the gene coding for tetracycline resistance, two sites lie within the tetracycline promoter and three within the β-lactamase gene.

pUC vectors

# Its name originated from University of California from where it was developed.

# 2.7 kb in size and possess:

# Gene for ampicillin resistance

# lacZ gene (coding for β-galactosidase gene) for blue/white  selection

# Origin of replication from pBR322

# Within the lacZ region there is a polylinker having unique RE  sites

Shuttle vectors

A shuttle vector is a vector (usually a plasmid) that can propagate in two  different host species. Thus the DNA fragment inserted into it can be  manipulated or tested in two different cell types.

The main advantage of using such a vector is that it can be manipulated  in E. coli and then introduced into a system which is more difficult or  slower to use (ex- yeast).

Thus a shuttle vector can propagate in eukaryotic and prokaryotic hosts and between different species of bacteria.

A shuttle vector is frequently used to make quickly multiple copies  (amplification) of the gene in E. coli. They can also be used for in vitro  experiments and modifications.

Yeast shuttle vector

Common yeast shuttle vectors contain:

# Bacterial origin of replication

# Bacterial selectable marker (antibiotic resistance)

# ARS (autonomously replicating sequence)

# Yeast selectable marker (LEU2)

# Yeast centromere

Major Limitation of Cloning in Plasmids

•Upper limit for insert DNA size is 12 kb

•Requires the preparation of “competent” host cells

•Inefficient for generating genomic libraries as overlapping  regions needed to place in proper sequence

•Preference for smaller clones to be transformed

•If it is an expression vector there are often limitations  regarding eukaryotic protein expression and post-translational modification

Phage Cloning Vectors

# Fragments up  to 23 kb can be may be accommodated by a phage  vector

# Lambda is most common phage

# Lambda phage is a virus that infects bacteria (E. coli)

# In 1971 Alan Campbell showed that the central third of its genome

was not required for lytic growth.

# People started to replace it with E. coli DNA (stuffer DNA)

# The stuffer fragment keeps the vector at a correct size and carries  marker genes that are removed when foreign DNA is inserted into the vector.

# Stuffer DNA contains a lacZ gene. When intact, beta-galactosidase  reacts with X-gal and the colonies turn blue.

# When the DNA segment replaces the stuffer the region, the lacZ gene  is missing, which codes for beta-galactosidase, no beta-galactosidase is formed, and the colonies are white.

Bacteriophage lambda (λ)

# Lambda genome is approximately 49 kb in size.

# Only 30 kb is  required for lytic growth.

# Thus, one could  clone 19 kb of  “foreign” DNA.

# Packaging efficiency  78%-100% of the lambda genome.

# Cos site: At the ends  short  (12bp)  ss-  complementary  region  “cohesive  or sticky” ends -- circulation after infection 

Lambda as a cloning vector

Insertional vectors:

—(clone into one or multiple restriction sites

—can only increase genome size by 5%

—size of foreign DNA insert depends on the original size of the phage  vector, about 5 to 11 kb

Replacement vectors (removing “stuffer”):

—can clone larger pieces of DNA, 8 to 24 kb (sufficient for many  eukaryotic genes)

Cosmid Cloning Vectors

Cosmids are plasmids that can be packaged into λ phage and they  combine essential elements of a plasmid and λ systems (cos sites).

Concatemer of unit length λ DNA molecules can be efficiently  packaged if cos sites are 37-54 kb apart.

Fragments from 30 to 46 kb can be accommodated by a 5 kb cosmid vector.

Cosmids are extracted from bacteria and mixed with restriction endonucleases.

Cleaved cosmids are mixed with foreign DNA that has been cleaved with the same endonuclease.

Recombinant cosmids are packaged into lambda capsids.

Recombinant cosmid is injected into the bacterial cell where the  rcosmid arranges into a circle and replicates as a plasmid. It can be maintained and recovered just as plasmids.

Yeast Artificial Chromosomes (YACs)

YACs are vectors constructed from yeast (Saccharomyces cerevisiae)  chromosomes to clone large DNA fragments

They are constructed as circular DNA molecule by assembling the  essential functions of natural yeast chromosomes, then splicing in a  fragment of foreign DNA.

This engineered chromosome is reinserted into a yeast cell to produce the YAC.

Specific sequences of YAC:

Telomeres: Located at the two ends of each chromosome. They have  evolved as a device to preserve the integrity of the ends of DNA  molecule, i.e. to protect the linear DNA from degradation by  nucleases

Centromere: Attachment site of the mitotic spindle fibers. They pull  one copy of each duplicated chromosome into each new daughter cell.

Origin of replication: Specific DNA sequence that allows DNA  replication machinery to assemble on the DNA.

Selectable markers: Allows easy selection of the yeast cells that  have taken up the YAC

RE sites: For insertion of the foreign DNA

Bacterial Artificial Chromosomes (BACs)

BAC is a cloning vector in E. coli developed as an alternative to YAC  vector for mapping and analysis of complex genomes.

BACs are maintained in E. coli as large single copy plasmid that  contains inserts of 50 – 350 kb with a high degree of stability.

A number of human and plant BAC libraries have been constructed.

Ex- Human, Arabidopsis, Rice, etc.

BAC system is based on the single-copy sex factor F of E. coli (~100 kb  circular ds DNA)

The synthetic BAC vectors (~7.5 kb, double-stranded) contains  replication origin OriS and gene repB of F plasmid for the initiation  and proper orientation of replication of BAC vector.

The parA and parB genes of F plasmid that ensure efficient segregation  of the F plasmid into the daughter E. coli cells after replication is also  incorporated into the BAC vector.

This vector also includes the following: 

     1. λcosN (single-stranded, complementary extensions of λ phage DNA for packaging dependent cleavage) 

          2. lox P sites (recognized by phage dependent recombinants) 

          3. Two cloning sites (HinDIII and BamHI) 

          4. Several G+C RE sites (SfiI, NotI, etc.) for potential excision of the inserts 

         5. Cloning sites are also flanked by T7 and SP6 promoters for generating RNA probes 

          6. Selectable marker genes for antibiotic resistance (CMR)

PAC

P1 Artificial Chromosome (a derivative of bacteriophage P1)

These vectors are constructed using DNA of P1 bacteriophages.

Can carry inserts 80-kb to 100-kb

These vectors contain essential replication components of P1phage  incorporated into a plasmid.

PAC was developed as a cloning vector by Nat Sternberg and  colleagues in the 1990s.