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Lecture 25: DNA Replication (Biochemical View)

Biochemistry

Learning Objectives

By the end of this lecture, students will be able to:

1. Describe the structure of DNA and the biochemical basis for semiconservative replication
2. Identify the enzymes and proteins involved in prokaryotic and eukaryotic DNA replication
3. Explain the mechanism of DNA polymerase, including its requirement for a primer and its 3' to 5' exonuclease proofreading activity
4. Describe the differences between leading and lagging strand synthesis, including the role of Okazaki fragments
5. Explain the end-replication problem and the role of telomerase
6. Describe the major DNA repair mechanisms and their clinical significance

Lecture Content

I. DNA Structure Review (Biochemical Perspective)

DNA is a double-stranded antiparallel helix held together by complementary base pairing: adenine pairs with thymine through 2 hydrogen bonds (A=T), and guanine pairs with cytosine through 3 hydrogen bonds (G≡C). The phosphodiester backbone links the 3'-OH of one nucleotide to the 5'-phosphate of the next, and the two strands run antiparallel, with one oriented 5' to 3' and the other 3' to 5'. The predominant B-form DNA is a right-handed helix with 10 base pairs per turn, a pitch of 3.4 nm, and both major and minor grooves that serve as protein recognition sites. Chargaff's rules -- the observation that %A = %T and %G = %C -- are a direct consequence of base pairing.

DNA stability arises from hydrogen bonds between bases, base-stacking interactions (van der Waals forces between the planar aromatic rings), and the hydrophobic effect. Denaturation (melting) -- the separation of the two strands -- can be induced by heat, alkali, or low ionic strength. The melting temperature (Tm) is higher for GC-rich DNA because of the three hydrogen bonds per GC pair. Denaturation produces a characteristic hyperchromic effect, in which absorbance at 260 nm increases as the bases become unstacked.

II. Overview of DNA Replication

DNA replication is semiconservative: each daughter duplex contains one parental strand and one newly synthesized strand, as demonstrated by the Meselson-Stahl experiment. Replication is bidirectional, proceeding in both directions from the origin of replication, and semi-discontinuous, with the leading strand synthesized continuously and the lagging strand synthesized in short fragments called Okazaki fragments. Prokaryotes have a single circular chromosome with a single origin of replication (oriC), while eukaryotes have multiple linear chromosomes with multiple origins of replication to allow their large genomes to be replicated within a reasonable time. Replication occurs during the S phase of the cell cycle.

III. Enzymology of DNA Replication

A. DNA Polymerases — The Central Enzyme

All DNA polymerases share several fundamental properties. They synthesize DNA in the 5' to 3' direction only, adding nucleotides to the 3'-OH of the growing chain. They require a template strand (read 3' to 5') and a primer with a free 3'-OH (they cannot initiate synthesis de novo); the primer is a short RNA sequence laid down by primase. They use dNTPs (dATP, dGTP, dCTP, dTTP) as substrates, releasing pyrophosphate that is hydrolyzed by pyrophosphatase to drive the reaction forward. DNA polymerases achieve extraordinarily high fidelity through three mechanisms: base selection by the enzyme (approximately 1 error per 10^4 to 10^5), 3' to 5' exonuclease proofreading (improving accuracy roughly 100-fold), and post-replication mismatch repair (improving accuracy roughly 1000-fold), for an overall error rate of approximately 1 mistake per 10^9 to 10^10 base pairs.

Prokaryotic DNA polymerases in E. coli include DNA Pol I, which removes RNA primers using its 5' to 3' exonuclease activity, fills the resulting gaps with DNA, possesses 3' to 5' proofreading, and participates in DNA repair. DNA Pol III is the main replicative polymerase, functioning as a holoenzyme with high processivity conferred by the beta-clamp sliding clamp and possessing 3' to 5' proofreading capability.

Eukaryotic DNA polymerases include DNA Pol alpha/primase, which initiates replication by synthesizing a short RNA primer followed by a short DNA extension but lacks proofreading activity. DNA Pol epsilon carries out leading strand synthesis with 3' to 5' proofreading. DNA Pol delta performs lagging strand synthesis with 3' to 5' proofreading and uses PCNA as its sliding clamp. DNA Pol gamma replicates mitochondrial DNA. DNA Pol beta fills gaps during base excision repair.

B. Other Replication Proteins

<image>A detailed diagram of the replication fork showing all major proteins and their functions. The parental DNA double helix is shown unwinding at the fork. Helicase (hexameric ring) is at the fork junction, unwinding the strands. SSB/RPA proteins coat the single-stranded template regions. Topoisomerase is shown ahead of the fork relieving supercoils. On the leading strand: DNA polymerase (Pol epsilon or Pol III) synthesizes continuously in the 5'->3' direction with the sliding clamp (PCNA/beta-clamp) attached. On the lagging strand: primase synthesizes short RNA primers, DNA polymerase (Pol delta or Pol III) extends each primer as an Okazaki fragment, RNase H removes RNA primers, DNA polymerase fills the gaps, and DNA ligase seals the nicks. The lagging strand template is shown looping back to keep both polymerases at the fork (trombone model). The direction of fork movement and the 5'->3' polarity of synthesis on both strands are clearly labeled.</image>

IV. Mechanism of Replication

A. Initiation

In prokaryotes, DnaA protein binds to the oriC sequence, which is AT-rich (and therefore easier to melt due to only 2 hydrogen bonds per A-T pair). DnaA-ATP melts the DNA, and DnaB helicase is loaded with assistance from DnaC. In eukaryotes, the Origin Recognition Complex (ORC) binds origins during G1 phase, and the MCM helicase is loaded in a process called licensing. MCM helicase is then activated by CDK and DDK kinases at the onset of S phase. Each origin fires only once per cell cycle, enforced by the licensing mechanism that prevents re-replication.

B. Elongation

On the leading strand, DNA polymerase synthesizes continuously in the 5' to 3' direction (the same direction as fork movement) after a single initial RNA primer. On the lagging strand, synthesis is discontinuous, producing Okazaki fragments (100 to 200 nucleotides in eukaryotes; 1000 to 2000 nucleotides in prokaryotes). Each fragment requires a new RNA primer from primase. DNA polymerase extends from each primer until it encounters the previous fragment. RNA primers are then removed (by RNase H in eukaryotes; by Pol I's 5' to 3' exonuclease in E. coli), gaps are filled with DNA, and DNA ligase seals the remaining nicks to create a continuous strand.

C. Termination

In prokaryotes, replication forks meet at the ter region, where the Tus protein blocks helicase progression. In eukaryotes, forks from adjacent origins converge, and topoisomerase II resolves concatenated daughter molecules.

V. Telomeres and the End-Replication Problem

The end-replication problem arises because the lagging strand cannot replicate the very end of a linear chromosome. When the terminal RNA primer is removed, there is no upstream 3'-OH available to fill the gap, so each round of replication shortens the chromosome by approximately 50 to 200 base pairs at each end. Telomeres are repetitive sequences at chromosome ends (TTAGGG repeats in humans, extending 5 to 15 kb) that serve as disposable buffers protecting coding sequences from erosion. They form protective T-loops stabilized by the shelterin protein complex.

Telomerase is a specialized reverse transcriptase (a ribonucleoprotein) that solves the end-replication problem. It contains an RNA template component (TERC/hTR) encoding the telomere repeat sequence and a catalytic subunit (TERT/hTERT) with reverse transcriptase activity. Telomerase extends the 3' end of the G-rich strand, after which conventional DNA polymerase fills in the complementary C-rich strand. Telomerase is active in germ cells, stem cells, and most cancer cells (approximately 85 to 90% of cancers reactivate telomerase). It is not active in most somatic cells, leading to progressive telomere shortening with each division, eventually reaching the Hayflick limit and triggering cellular senescence.

VI. DNA Repair Mechanisms

A. Proofreading (3'->5' Exonuclease)

Proofreading is built into replicative DNA polymerases. When a mismatched base is incorporated, the polymerase detects the distortion, the 3' to 5' exonuclease activity removes the incorrect nucleotide from the 3' end, and the correct nucleotide is then inserted.

B. Mismatch Repair (MMR)

Mismatch repair corrects errors that escape proofreading after replication is complete. The system must distinguish the newly synthesized strand (which contains the error) from the parental strand. In E. coli, the new strand lacks methylation at GATC sites; in eukaryotes, strand breaks or nicks likely serve as the discrimination signal. MutS recognizes the mismatch, MutL coordinates the repair process, and the mismatched segment of the new strand is excised and resynthesized. The eukaryotic homologs are MSH2, MSH6, MLH1, and PMS2. Defects in mismatch repair cause Lynch syndrome (hereditary nonpolyposis colorectal cancer, HNPCC), an autosomal dominant condition featuring microsatellite instability.

C. Base Excision Repair (BER)

BER repairs small, non-bulky base lesions such as deamination, oxidation, and alkylation products. A DNA glycosylase specific for the damaged base recognizes and removes it, creating an AP site (apurinic/apyrimidinic). AP endonuclease then cleaves the backbone at the AP site. DNA Pol beta fills the single-nucleotide gap, and DNA ligase seals the nick. A classic example is the repair of uracil in DNA (arising from spontaneous deamination of cytosine) by uracil-DNA glycosylase.

D. Nucleotide Excision Repair (NER)

NER repairs bulky, helix-distorting lesions such as UV-induced thymine dimers and benzo[a]pyrene adducts. The system recognizes the helical distortion, excises a segment of approximately 25 to 30 nucleotides containing the damage, and DNA polymerase fills the gap while ligase seals the nick. Defects in NER cause xeroderma pigmentosum (XP), an autosomal recessive disorder with extreme UV sensitivity and greater than 1000-fold increased skin cancer risk, resulting from mutations in XPA through XPG genes.

E. Double-Strand Break Repair

Double-strand breaks are the most dangerous form of DNA damage. Two repair pathways exist. Homologous recombination (HR) uses the sister chromatid as a template and is error-free; it operates mainly in S and G2 phases and involves BRCA1, BRCA2, and RAD51. Mutations in BRCA1 or BRCA2 increase the risk of breast and ovarian cancer. Non-homologous end joining (NHEJ) directly ligates the broken ends without a template and is therefore error-prone (nucleotides may be lost); it is available in all cell cycle phases and involves Ku70/Ku80, DNA-PKcs, XRCC4, and Ligase IV.

<image>A comparison panel of the four major DNA repair mechanisms. Panel A (Base Excision Repair): A damaged base (e.g., uracil from cytosine deamination) is shown in a DNA strand. DNA glycosylase removes the base creating an AP site. AP endonuclease cuts the backbone. DNA Pol beta inserts the correct nucleotide. Ligase seals. Panel B (Nucleotide Excision Repair): A bulky lesion (thymine dimer from UV) distorts the helix. The damaged region is recognized, dual incisions are made flanking the lesion, a 25-30 nt segment is removed, DNA polymerase fills the gap, and ligase seals. Panel C (Mismatch Repair): A G-T mismatch after replication is shown. MutS recognizes the mismatch, MutL is recruited, the newly synthesized strand is identified and excised around the mismatch, and the gap is resynthesized correctly. Panel D (Double-Strand Break Repair): Two pathways are shown — homologous recombination (using sister chromatid, error-free, involving BRCA1/2 and RAD51) and NHEJ (direct ligation by Ku proteins, error-prone). Clinical associations are noted: XP for NER defects, Lynch syndrome for MMR defects, BRCA mutations for HR defects.</image>

VII. Pharmacological Targets in DNA Replication

Several classes of drugs target the replication machinery. Fluoroquinolones (such as ciprofloxacin) inhibit bacterial DNA gyrase (topoisomerase II) and topoisomerase IV. Acyclovir is a nucleoside analog that acts as a chain terminator after phosphorylation by viral thymidine kinase in herpes-infected cells, because it lacks a 3'-OH. AZT (zidovudine) is a nucleoside reverse transcriptase inhibitor used in HIV treatment that also functions as a chain terminator. Cytarabine (Ara-C) inhibits DNA polymerase and is used in leukemia treatment. Topotecan and irinotecan inhibit eukaryotic topoisomerase I, while etoposide and doxorubicin inhibit eukaryotic topoisomerase II; all are anticancer agents. Methotrexate and 5-FU inhibit thymidylate synthesis and reduce dTTP pools, as discussed in Lecture 23.

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