Origins of Replication

Getting Started

• DNA replication starts at specific locations called origins of replication
• These are short DNA segments with a unique nucleotide sequence
• E. coli chromosomes, like other bacterial chromosomes, are circular and possess a single origin
• Proteins that begin DNA replication identify and bind to this sequence
• The proteins separate the two strands and create a replication bubble
• DNA replication happens in both directions until the entire molecule has been copied
• Eukaryotic chromosomes have hundreds or thousands of replication origins
• Replication bubbles merge, speeding up the copying process

Origins of Replication

• In E. coli and additional bacterial cells, replication begins at a single origin
• Parental strands separate at the origin, creating a replication bubble with two forks
• Replication continues in both directions until the forks meet on the opposite side, resulting in two daughter DNA molecules
• Eukaryotic DNA replication proceeds in both directions from each origin
• A replication fork is at each end of the replication bubble
• At the fork, the parental DNA strands are unwound
• Enzymes called helicases unwind the double helix at the replication forks
• They separate the two parental strands and make them available as template strands
• Single-strand binding proteins attach to unpaired DNA strands
• They prevent the strands from re-pairing
• Topoisomerase relieves the strain caused by the untwisting of the double helix
• It breaks, swivels, and rejoins DNA strands

Synthesizing a New DNA Strand

• Unwound parental DNA strands function as templates for synthesizing new, complementary DNA strands
• DNA polymerases need a pre-existing chain to start adding DNA nucleotides
• A primer, a short RNA segment, is created during DNA synthesis
• The primer is synthesized by the enzyme primase
• Primase initiates a complementary RNA chain by adding RNA nucleotides one at a time
• A completed primer is a sequence of 5-10 nucleotides that are base-paired to the template strand
• The new DNA strand starts from the 3' end of the RNA primer
• DNA polymerases are enzymes that synthesize new DNA by adding nucleotides to the 3' end
• E. coli has several DNA polymerases, but DNA polymerase III and DNA polymerase I are primarily responsible for DNA replication
• Eukaryotes have at least 11 different DNA polymerases
• Most DNA polymerases need a primer and a DNA template strand
• In E. coli, DNA polymerase III (DNA pol III) adds a DNA nucleotide to the RNA primer
• It then continues adding DNA nucleotides complementary to the parental DNA template strand
• Elongation occurs at a rate of approximately 500 nucleotides per second in bacteria
• Elongation occurs at a rate of approximately 50 nucleotides per second for human cells
• Nucleotides added to a growing DNA strand consist of a sugar, a base, and three phosphate groups
• ATP (adenosine triphosphate) is a molecule consisting of a sugar, a base, and three phosphate groups
• The only difference between ATP and dATP is the sugar component; dATP has deoxyribose, whereas ATP has ribose
• The nucleotides used for DNA synthesis are chemically reactive because of their triphosphate tails
• DNA polymerase catalyzes the addition of monomers using a condensation reaction, in which two phosphate groups are lost as pyrophosphate (PPi)
• Hydrolysis of pyrophosphate into two inorganic phosphate molecules (Pi) is a coupled exergonic reaction that helps drive polymerization

Antiparallel Elongation

• DNA strands have directionality, with two distinct ends
• The two DNA strands in a double helix are antiparallel
• New strands formed during DNA replication must also be antiparallel to their template strands
• DNA polymerases can only add nucleotides to the free 3' end of a primer or growing DNA strand, meaning that a new DNA strand elongates only in the 5' → 3' direction
• Along one template strand, DNA polymerase III creates a complementary strand continuously in the mandatory 5' → 3' direction
• This new strand is called the leading strand
• Only one primer is needed for DNA pol III to synthesize the entire leading strand
• To elongate the other new strand in the mandatory 5' → 3' direction, DNA pol III works along the other template strand away from the replication fork
• The DNA strand elongating in this direction is called the lagging strand
• The lagging strand is synthesized discontinuously, in a series of segments
• These segments are called Okazaki fragments
• Okazaki fragments are 1,000-2,000 nucleotides long in E. coli and 100-200 nucleotides long in eukaryotes

Synthesis of Leading Strand During DNA Replication

• DNA polymerase III (DNA pol III) is associated with a protein sliding clamp, which moves it along the DNA template strand
• After an RNA primer is made, DNA pol III starts to synthesize the leading strand
• The leading strand is elongated continuously in the 5' → 3' direction

Synthesis of Lagging Strand

• Primase joins RNA nucleotides into the first primer for the lagging strand
• DNA pol III adds DNA nucleotides to the primer, forming Okazaki fragment 1
• After reaching the next RNA primer, DNA pol III detaches
• After the last addition, the backbone is left with a free 3' end
• DNA ligase forms a bond between the newest DNA and the DNA of fragment 1
• The lagging strand in this region is now complete

The DNA Replication Complex

• The lagging strand's name is due to a slight delay between it and the leading strand
• The new DNA Replication Complex is a collection of various proteins
• Primase acts as molecular break, coordinating the rate of leading and lagging strand synthesis
• The DNA moves through the complex
• In eukaryotic cells, multiple complexes are grouped into "factories”
• They may be anchored to the nuclear matrix
• Two DNA polymerase molecules are present, "reeling in" parental DNA and extruding newly made daughter DNA in a "trombone model"
• In the trombone model, the lagging strand is looped back through the complex
• It is unresolved whether the complex moves along the DNA or whether the DNA moves through the complex

Bacterial DNA Replication

• Molecules of single-strand binding protein stabilize the unwound template strands
• The leading strand is synthesized continuously in the 5' to 3' direction by DNA pol III
• Primase begins synthesis of the RNA primer for the fifth Okazaki fragment
• DNA pol III is completing synthesis of fragment 4
• When it reaches the RNA primer on fragment 3, it will detach and begin adding DNA nucleotides to the 3' end of the fragment 5 primer in the replication fork
• DNA pol I removes the primer from the 5' end of fragment 2
• It is replaced by DNA nucleotides added one by one to the 3' end of fragment 3
• DNA ligase joins the 3' end of fragment 2 to the 5' end of fragment 1

Bacterial DNA Replication Proteins and Their Functions

Proofreading and Repairing DNA

• DNA replication accuracy does not solely rely on base pairing specificity
• Initial pairing errors occur at a rate of one in 105
• Errors in the completed DNA molecule amount to only one in 1010 nucleotides
• DNA polymerases proofread each nucleotide against its template as soon as it is covalently bonded
• If there's an incorrectly paired nucleotide, the polymerase removes the nucleotide and then resumes synthesis
• In mismatch repair, other enzymes remove and replace incorrectly paired nucleotides that have resulted from replication errors
• Maintenance requires frequent repair of various kinds of damage to existing DNA
• DNA molecules are constantly subjected to potentially harmful chemical and physical agents
• Spontaneous chemical changes to DNA bases must be corrected before becoming permanent changes (mutations)
• Each cell continuously monitors and repairs its genetic material
• Most cellular systems for repairing incorrectly paired nucleotides use a mechanism that takes advantage of the base-paired structure of DNA
• One system involves cutting out the section of damage (excision) by a DNA-cutting enzyme (a nuclease)
• A DNA polymerase and DNA ligase then fill in any gaps
• One repair system is called nucleotide excision repair

Nucleotide Excision Repair of DNA Damage

• Teams of enzymes detect and repair damaged DNA, such as thymine dimers (often caused by ultraviolet radiation)
• A nuclease enzyme cuts the damaged DNA strand at two points, and the damaged section is removed
• Repair synthesis by a DNA polymerase fills in the missing nucleotides, using the undamaged strand as a template
• DNA ligase seals the free end of the new DNA to the old DNA, making the strand complete

Evolutionary Significance of Altered DNA Nucleotides

• Correct DNA duplication and damage repair are essential for the organism and the next generation
• The error rate after proofreading is extremely low, but rare mistakes do slip through
• Once a mismatched nucleotide, the new sequence change is permanent in all daughter cells
• Mutations can change the phenotype and be passed on from generation to generation
• Mutations have small or no effect, or can be harmful or beneficial
• Mutations are the original source of the variation on which natural selection operates
• New proteins contribute to different phenotypes

Replicating the Ends of DNA Molecules

• Linear DNA replicates and has 5' ends of daughter DNA strands
• There is no 3' end of a pre-existing polynucleotide for DNA polymerase to add onto to
• Even if an Okazaki fragment can be started with an RNA primer hydrogen-bonded to the end of the template strand, once that primer is removed, it cannot be replaced with DNA because there is no 3'end available
• Circular chromosomes don’t need ends
• Eukaryotic chromosomal DNA molecules have special nucleotide sequences called telomeres at their ends
• Telomeres do not contain genes; instead, the DNA consists of multiple repeats of one short nucleotide sequence
• Telomeres enable proteins from activating to prevent DNA damage
• Also, act as a buffer zone against gene shortening

Telomeres

• Shortening of telomeres is connected to the aging process of certain tissues and organisms
• Telomerase catalyz