DNA Replication

Getting Started

• Chromosomal DNA replication commences at specific sites known as origins of replication.

• These sites consist of short DNA stretches with specific nucleotide sequences.

• The E. coli chromosome is circular with a single origin.

• Proteins required for this process recognize the origin, attach to the DNA, and separate the strands.

• Separation of DNA leads to a replication bubble.

• DNA replication proceeds bidirectionally until the entire molecule has been replicated.

• Eukaryotic chromosomes have hundreds to thousands of replication origins.

• Multiple replication bubbles form and fuse to expedite copying.

• Eukaryotic and bacterial DNA replication proceeds in both directions from each origin.

• A replication fork is located at each end of the replication bubble.

• Helicases - enzymes, untwist the double helix at the replication forks, separating the parent strands and making available the template strands.

• Single-strand binding proteins - proteins that then bind to unpaired strands to prevent re-pairing.

• Topoisomerase prevents over-twisting that results from the separation of the double helix, through relieving this strain by breaking, swiveling, and rejoining DNA strands.

Synthesizing a New DNA Strand

• During synthesis of new strands, DNA polymerase can only add nucleotides to an already existing chain.
• DNA synthesis initiation requires a pre-existing chain, produced as a short stretch of RNA called a primer.
• Primase synthesizes the RNA primer using the parental DNA strand as a template.
• The completed primer consists of five to ten nucleotides and is base-paired to the template strand.
• The new DNA strand starts from the 3' end of the RNA primer.
• DNA polymerase catalyzes new DNA synthesis by adding nucleotides to the 3' end of a pre-existing chain.
• DNA polymerase III and Polymerase I are major enzymes in E. coli DNA replication.
• At least 11 different types of DNA polymerase have been discovered in eukaryotes.
• Most DNA polymerases require a primer and a DNA template strand to line up complementary DNA nucleotides.
• In E. coli, DNA polymerase III adds a DNA nucleotide to the RNA primer, then adds nucleotides to the new strand.
• Elongation occurs at approximately 500 nucleotides per second in bacteria and 50 per second in human cells.
• Each nucleotide added to a DNA strand contains a sugar, a base, and three phosphate groups.
• ATP has deoxyribose as its sugar component, while ATP has ribose.
• Triphosphate tails of nucleotides used for DNA synthesis are unstable and chemically reactive.
• During each monomer addition, DNA polymerase catalyzes a condensation reaction and two phosphate groups are lost as pyrophosphate (PPi).
• Subsequent pyrophosphate hydrolysis (PPi to 2Pi) drives polymerization.

Antiparallel Elongation

• Two DNA strand ends are different, giving each strand directionality.

• Two DNA strands in a double helix run antiparallel, meaning they are oriented in opposite directions.

• New DNA strands are also antiparallel to their template strands.

• Because of DNA polymerase structure, nucleotides can only be added to the free 3' end of a primer or growing DNA strand.

• A new DNA strand can elongate only in the 5' to 3' direction.

• Along one template strand, DNA polymerase III synthesizes a continuous complementary strand in the 5' to 3' direction.

• The DNA polymerase remains in the replication fork on the template strand as it adds nucleotides to the new complementary strand.

• The DNA produced this way is the leading strand.

• Only one primer is required to synthesize the entire leading strand by DNA pol.

• Elongation of the other new strand of DNA in the 5' to 3' direction requires DNA pol working along the template strand away from the replication fork.

• The DNA made this way is called the lagging strand.

• The lagging strand is synthesized discontinuously as Okazaki fragments.

• Okazaki fragments are about 1,000-2,000 nucleotides long in E. coli and 100-200 nucleotides long in eukaryotes.

• Synthesis of the leading and lagging strands occurs concurrently at the same rate with the lagging strand delayed slightly.

• Lagging strand synthesis is delayed as each fragment cannot start until sufficient template has been exposed.

• The proteins and enzymes that participate in the process form a "DNA replication machine."

• Primase acts as a molecular brake, coordinating primer placement and replication rates on the leading and lagging strands.

• The DNA replication complex may remain stationary as DNA moves through it.

• In eukaryotes, multiple complex copies, may be anchored to the nuclear matrix.

• A trombone model posits that two DNA polymerase molecules reel in parental DNA and extrude new daughter DNA molecules.

• Looping the lagging strand back through the complex facilitates this process.

Proofreading and Repairing DNA

• DNA replication accuracy is not due only to the specificity of based pairing.
• Initial pairing errors between incoming nucleotides and template strands occur at a rate of 1 in 10^5 nucleotides.
• Final error rate in completed DNA is one in 10^10 nucleotides.
• DNA polymerases proofread nucleotides as soon as added to the growing strand which makes replication more accurate by a factor of 100,000.
• Mismatched nucleotides are removed and replaced before synthesis resumes.
• Mismatch repair involves enzymes removing and replacing incorrectly paired nucleotides resulting from replication errors.
• Imperfectly paired or altered nucleotides can occur after replication.
• Maintaining genetic information requires damage repair of existing DNA
• DNA molecules are constantly exposed to harmful chemicals/physical agents, or spontaneous chemical changes occur under normal conditions.
• Continuous monitoring and repair of genetic material is performed continuously.
• Many different DNA repair enzymes have evolved because damaged repair of damaged DNA is essential for survival.
• Nearly 100 E. coli repair enzymes are identified, with about 170 in humans..
• Cellular systems for repairing incorrectly paired nucleotides work by removing the damaged segment using a nuclease
• The resulting gap is filled with nucleotides using the undamaged strand as a template.
• DNA polymerase and DNA ligase are involved in filling the gaps.

Evolutionary Significance of Altered DNA Nucleotides

• Genome replication and DNA damage repair ensures organism function and accurate genome inheritance.
• A low mutation rate has resulted in new proteins that contribute to different phenotypes
• The error rate is reduced to 1 in 10^10 which is lower than would be expected from simple base-pairing, after proof-reading and repair.
• A permanent change in the DNA sequence that is replicated is a mutation.
• Mutations can change an organism's phenotype, and are passed on in gametes from generation to generation.
• A low mutation rate and complete fidelity of DNA replicaiton results in new proteins contributing to different phenotypes.
• Over extended time periods, it leads to new species and the rich diversity on Earth.

Replicating the Ends of DNA Molecules

• Replication machinery cannot complete linear eukaryotic chromosome 5' ends.
• This is because DNA polymerase can only add to an existing 3' end of a polynucleotide.
• Even started with an primer hydrogen-bonded to the template strand, once removed, no additional nucleotides can be added to because there’s no 3’ end available for nucleotide addition.
• This leads to shorted DNA ends after repeated replication rounds.
• Shortening of DNA does not occur in nearly all prokaryotes because they have a circular chromosome,
• Eukaryotic chromosomal DNA molecules contain nucleotide sequences termed telomeres at their ends.
• Telomeres consist of multiple repetitions of a short nucleotide sequence.
• Telomeres have 2 functions, they activate the cell’s systems for monitoring DNA damage and they protect from shortening genes.
• The enzyme telomerase catalyzes lengthening of telomeres, restoring lost length from replication and using its molecule as at template to artificially "extend" the leading strand.
• Telomerase is not active in most human somatic cells.