DNA Replication

The Biochemical Reactions

• DNA replication begins with the "unzipping" of the parent molecule as the hydrogen bonds between the base pairs are broken.
• Once exposed, the sequence of bases on each of the separated strands serves as a template to guide the insertion of a complementary set of bases on the strand being synthesized.
• The new strands are assembled from deoxynucleoside triphosphates.
• Each incoming nucleotide is covalently linked to the "free" 3' carbon atom on the pentose (figure) as
• the second and third phosphates are removed together as a molecule of pyrophosphate (PPi).
• The nucleotides are assembled in the order that complements the order of bases on the strand serving as the template.
• Thus each C on the template guides the insertion of a G on the new strand, each G a C, and so on.
• When the process is complete, two DNA molecules have been formed identical to each other and to the parent molecule.

The Enzymes

• A portion of the double helix is unwound by a helicase.
• A molecule of a DNA polymerase binds to one strand of the DNA and begins moving along it in the 3' to 5' direction, using it as a template for assembling a leading strand of nucleotides and reforming a double helix. In eukaryotes, this molecule is called DNA polymerase delta (δ).
• Because DNA synthesis can only occur 5' to 3', a molecule of a second type of DNA polymerase (epsilon, ε, in eukaryotes) binds to the other template strand as the double helix opens. This molecule must synthesize discontinuous segments of polynucleotides (called Okazaki fragments). Another enzyme, DNA ligase I then stitches these together into the lagging strand.

DNA Replication is Semiconservative

When the replication process is complete, two DNA molecules — identical to each other and identical to the original — have been produced. Each strand of the original molecule has

• remained intact as it served as the template for the synthesis of
• a complementary strand.

This mode of replication is described as semi-conservative: one-half of each new molecule of DNA is old; one-half new. Watson and Crick had suggested that this was the way the DNA would turn out to be replicated. Proof of the model came from the experiments of Meselson and Stahl. [Link to them.]

Speed of Replication

Bacteria

The single molecule of DNA that is the E. coli genome contains 4.7 x 10⁶ nucleotide pairs. DNA replication begins at a single, fixed location in this molecule, the replication origin, proceeds at about 1000 nucleotides per second, and thus is done in no more than 40 minutes. And thanks to the precision of the process (which includes a "proof-reading" function), the job is done with only about one incorrect nucleotide for every 10⁹ nucleotides inserted. In other words, more often than not, the E. coli genome (4.7 x 10⁶) is copied without error!

Eukaryotes

The average human chromosome contains 150 x 10⁶ nucleotide pairs which are copied at about 50 base pairs per second. The process would take a month (rather than the hour it actually does) but for the fact that there are many places on the eukaryotic chromosome where replication can begin. Replication begins at some replication origins earlier in S phase than at others, but the process is completed for all by the end of S phase. As replication nears completion, "bubbles" of newly replicated DNA meet and fuse, finally forming two new molecules.

Control of Replication

With their multiple origins, how does the eukaryotic cell know which origins have been already replicated and which still await replication?
An observation: When a cell in G₂ of the cell cycle is fused with a cell in S phase, the DNA of the G₂ nucleus does not begin replicating again even though replication is proceeding normally in the S-phase nucleus. Not until mitosis is completed, can freshly-synthesized DNA be replicated again.
Two control mechanisms have been identified — one positive and one negative. This redundancy probably reflects the crucial importance of precise replication to the integrity of the genome.

Licensing: positive control of replication

In order to be replicated, each origin of replication must be bound by:

• an Origin Recognition Complex of proteins (ORC). These remain on the DNA throughout the process.
• Accessory proteins called licensing factors. These accumulate in the nucleus during G₁ of the cell cycle. They include:
  • Cdc-6 and Cdt-1, which bind to the ORC and are essential for coating the DNA with
  • MCM proteins. Only DNA coated with MCM proteins (there are 6 of them) can be replicated.

Once replication begins in S phase,

• Cdc-6 and Cdt-1 leave the ORCs (the latter by ubiquination and destruction in proteasomes).
• The MCM proteins leave in front of the advancing replication fork.

Geminin: negative control of replication

G₂ nuclei also contain at least one protein — called geminin — that prevents assembly of MCM proteins on freshly-synthesized DNA (probably by blocking the actions of Cdt1).
As the cell completes mitosis, geminin is degraded so the DNA of the two daughter cells will be able to respond to licensing factors and be able to replicate their DNA at the next S phase.

DNA Translation

Translation occurs in the cytoplasm where the ribosomes are located. Ribosomes are made of a small and large subunit which surround the mRNA.

Transfer RNA (tRNA) molecules are 75 - 95 nucleotides long and have four arms and three loops. True to its name, tRNA transfers amino acids to the site of the growing protein chain (polypeptide). Each tRNA molecule (in red below) recognises a specific, three base-pair mRNA code or codon (the DNA form of a codon is called a triplet and the sequence on the tRNA is called an anticodon). Since there are three bases and four possible nucleotides, there can be up to 64 (4x4x4) possible tRNA molecules. Three of these tRNA molecules recognise "stop" or termination codons which have been named amber (UAG), opal (UGA) and ochre (UAA).

The codon indicates which amino acid is to be added and the amino acid is attached to the tRNA molecule at the acceptor arm. As we can see from the table below, most amino acids are represented by more than one codon. This means that the expected protein can still be synthesised, even when a degree of mutation occurs in the DNA or mRNA.

There are 20 essential amino acids, however they can be combined in any order, just like the four nucleotides. This permits the production of the many different proteins which let organisms grow and function.

Name	1-Letter Nickname	Triplet	3-Letter Nickname
Glycine	G	GGT,GGC,GGA,GGG	Gly
Alanine	A	GCT,GCC,GCA,GCG	Ala
Valine	V	GTT,GTC,GTA,GTG	Val
Leucine	L	TTG,TTA,CTT,CTC,CTA,CTG	Leu
Isoleucine	I	ATT,ATC,ATA	Ileu
Serine	S	TCT,TCC,TCA,TCG,AGT,AGC	Ser
Threonine	T	ACT,ACC,ACA,ACG	Thr
Cysteine	C	TGT,TGC	Cys
Methionine	M	ATG	Met
Glutamic Acid	E	GAA,GAG	Glu
Aspartic Acid	D	GAT,GAC,AAT,AAC	Asp
Lysine	K	AAA,AAG	Lys
Arginine	R	CGT,CGC,CGA,CGG,AGA,AGG	Arg
Asparagine	N	AAT,AAC	Asn
Glutamine	Q	GAA,GAG	Gln
Phenylalanine	F	TTT,TTC	Phe
Tyrosine	Y	TAT, TAC	Tyr
Tryptophan	W	TGG	Trp
Unknown	X		xxx
Proline	P	CCT,CCC,CCA,CCG	Pro
Terminator	*	TAA,TAG,TGA	End

INITIATION

When the large ribosmal subunit, small ribosomal subunit, mRNA and the tRNA carrying a methionine come together in the cytoplasm, the ribosome becomes active and the synthesis of a polypeptide, or "translation", is initiated. The AUG codon binds at the protein binding site (P) of the ribosome and AUG is always the first codon of an mRNA.

The next complementary tRNA will bind at the attachment binding site (A) of the ribosome. The adjacent amino acids are then joined by a peptide bond via a peptidase enzyme. Thus the polypeptide chain begins to grow and as it does, it is passed to the next tRNA currently occupying the A site.

ELONGATION

The ribosome then moves 1 codon down the mRNA in a 5' to 3' direction. This is achieved by a translocase enzyme. As the process of ribosome translocation continues, the "old" tRNA is released to bind another amino acid and go in search of a new codon. The binding of a new aminoacid is mediated by an enzyme called amino-acyl-tRNA synthase

TERMINATION

The process continues along the mRNA until a stop codon is reached. While there is no tRNA for a stop codon, there is an enzyme called release factor which cleaves the polypeptide chain resulting in a new protein.

Finally, the entire complex is disrupted, the ribosome separates and the mRNA is released to be used again or degraded. Translation occurs at multiple sites along an mRNA so that many ribosomes can be seen by electron microscopy bound to a single mRNA strand with many polypeptide chains forming from each.

Not only do different sequences make different proteins, but slight sequence changes can radically change the shape of a protein. The shape or structure of the protein is essential for its correct function e.g. as an enzyme or an ion channel embedded in a cell membrane.

Introduction to DNA Structure

Components of DNA

DNA is a polymer. The monomer units of DNA are nucleotides, and the polymer is known as a "polynucleotide." Each nucleotide consists of a 5-carbon sugar (deoxyribose), a nitrogen containing base attached to the sugar, and a phosphate group. There are four different types of nucleotides found in DNA, differing only in the nitrogenous base. The four nucleotides are given one letter abbreviations as shorthand for the four bases.

• A is for adenine
• G is for guanine
• C is for cytosine
• T is for thymine

Purine Bases

Adenine and guanine are purines. Purines are the larger of the two types of bases found in DNA. Structures are shown below:

Structure of A and G

The 9 atoms that make up the fused rings (5 carbon, 4 nitrogen) are numbered 1-9. All ring atoms lie in the same plane.

Pyrimidine Bases

Cytosine and thymine are pyrimidines. The 6 stoms (4 carbon, 2 nitrogen) are numbered 1-6. Like purines, all pyrimidine ring atoms lie in the same plane.

Structure of C and T

Deoxyribose Sugar

The deoxyribose sugar of the DNA backbone has 5 carbons and 3 oxygens. The carbon atoms are numbered 1', 2', 3', 4', and 5' to distinguish from the numbering of the atoms of the purine and pyrmidine rings. The hydroxyl groups on the 5'- and 3'- carbons link to the phosphate groups to form the DNA backbone. Deoxyribose lacks an hydroxyl group at the 2'-position when compared to ribose, the sugar component of RNA.

Structure of deoxyribose

Nucleosides

A nucleoside is one of the four DNA bases covalently attached to the C1' position of a sugar. The sugar in deoxynucleosides is 2'-deoxyribose. The sugar in ribonucleosides is ribose. Nucleosides differ from nucleotides in that they lack phosphate groups. The four different nucleosides of DNA are deoxyadenosine (dA), deoxyguanosine (dG), deoxycytosine (dC), and (deoxy)thymidine (dT, or T).

Structure of dA

In dA and dG, there is an "N-glycoside" bond between the sugar C1' and N9 of the purine.

Nucleotides

A nucleotide is a nucleoside with one or more phosphate groups covalently attached to the 3'- and/or 5'-hydroxyl group(s).

DNA Backbone

The DNA backbone is a polymer with an alternating sugar-phosphate sequence. The deoxyribose sugars are joined at both the 3'-hydroxyl and 5'-hydroxyl groups to phosphate groups in ester links, also known as "phosphodiester" bonds.

Example of DNA Backbone: 5'-d(CGAAT):

Features of the 5'-d(CGAAT) structure:

• Alternating backbone of deoxyribose and phosphodiester groups
• Chain has a direction (known as polarity), 5'- to 3'- from top to bottom
• Oxygens (red atoms) of phosphates are polar and negatively charged
• A, G, C, and T bases can extend away from chain, and stack atop each other
• Bases are hydrophobic

DNA Double Helix

DNA is a normally double stranded macromolecule. Two polynucleotide chains, held together by weak thermodynamic forces, form a DNA molecule.

Structure of DNA Double Helix

Features of the DNA Double Helix

• Two DNA strands form a helical spiral, winding around a helix axis in a right-handed spiral
• The two polynucleotide chains run in opposite directions
• The sugar-phosphate backbones of the two DNA strands wind around the helix axis like the railing of a sprial staircase
• The bases of the individual nucleotides are on the inside of the helix, stacked on top of each other like the steps of a spiral staircase.

Base Pairs

Within the DNA double helix, A forms 2 hydrogen bonds with T on the opposite strand, and G forms 3 hyrdorgen bonds with C on the opposite strand.

Example of dA-dT base pair as found within DNA double helix

Example of dG-dC base pair as found within DNA double helix

• dA-dT and dG-dC base pairs are the same length, and occupy the same space within a DNA double helix. Therefore the DNA molecule has a uniform diameter.
• dA-dT and dG-dC base pairs can occur in any order within DNA molecules

DNA Helix Axis

The helix axis is most apparent from a view directly down the axis. The sugar-phosphate backbone is on the outside of the helix where the polar phosphate groups (red and yellow atoms) can interact with the polar environment. The nitrogen (blue atoms) containing bases are inside, stacking perpendicular to the helix axis.

View down the helix axis

DNA extraction

DNA isolation is a routine procedure to collect DNA for subsequent molecular or forensic analysis. There are three basic and one optional steps in a DNA extraction:

1. Breaking the cells open, commonly referred to as cell disruption or cell lysis, to expose the DNA within. This is commonly achieved by grinding or sonicating the sample.
2. Removing membrane lipids by adding a detergent.
3. Removing proteins by adding a protease (optional but almost always done).
4. Precipitating the DNA with an alcohol — usually ice-cold ethanol or isopropanol. Since DNA is insoluble in these alcohols, it will aggregate together, giving a pellet upon centrifugation. This step also removes alcohol-soluble salt.

Refinements of the technique include adding a chelating agent to sequester divalent cations such as Mg²⁺ and Ca²⁺, which prevents enzymes like DNAse from degrading the DNA.

Cellular and histone proteins bound to the DNA can be removed either by adding a protease or by having precipitated the proteins with sodium or ammonium acetate, or extracted them with a phenol-chloroform mixture prior to the DNA-precipitation.

If desired, the DNA can be resolubilized in a slightly alkaline buffer or in ultra-pure water.

For the various methods, see Nucleic acid methods.

What is DNA?

Deoxyribonucleic acid (/diˌɒksiˌraɪbɵ.njuːˌkleɪ.ɨk ˈæsɪd/  ( listen)), or DNA, is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms, with the exception of some viruses. The main role of DNA molecules is the long-term storage of information. DNA is often compared to a set of blueprints, like a recipe or a code, since it contains the instructions needed to construct other components of cells, such as proteins and RNA molecules. The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in regulating the use of this genetic information.

DNA consists of two long polymers of simple units called nucleotides, with backbones made of sugars and phosphate groups joined by ester bonds. These two strands run in opposite directions to each other and are therefore anti-parallel. Attached to each sugar is one of four types of molecules called bases. It is the sequence of these four bases along the backbone that encodes information. This information is read using the genetic code, which specifies the sequence of the amino acids within proteins. The code is read by copying stretches of DNA into the related nucleic acid RNA, in a process called transcription.

Within cells, DNA is organized into long structures called chromosomes. These chromosomes are duplicated before cells divide, in a process called DNA replication. Eukaryotic organisms (animals, plants, fungi, and protists) store most of their DNA inside the cell nucleus and some of their DNA in organelles, such as mitochondria or chloroplasts. In contrast, prokaryotes (bacteria and archaea) store their DNA only in the cytoplasm. Within the chromosomes, chromatin proteins such as histones compact and organize DNA. These compact structures guide the interactions between DNA and other proteins, helping control which parts of the DNA are transcribed.