Replication Basics

Replication Basics

Mismatch repair further improves fidelity after

Despite proofreading, occasional mismatches escape locking before next polymerization step. Post-replicative mismatch repair scans daughter strand discriminated in E. coli by transient hemimethylation of GATC sites remaining unmethylated for minutes. MutS protein detects backbone distortion caused by mismatch, MutL coordinates endonuclease MutH cleavage of unmethylated nascent strand, and helicase plus exonucleases remove error-containing segment. Resynthesis by Pol III restores correct sequence using parental strand template. This pathway improves fidelity additional 100 to 1000-fold and explains hypermutator phenotype in MutSL deficient strains.

Ref: Alberts et al., Molecular Biology of the Cell, 7th ed., Chapter 5: Methyl-directed mismatch repair after replication

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Replication Basics

DNA polymerase cannot start synthesis de novo because it lacks

DNA polymerases catalyze attack of 3' hydroxyl on alpha-phosphate of incoming dNTP forming phosphodiester linkage. They cannot create that hydroxyl de novo nor initiate phosphodiester bond between two free dNTPs alone. Initiation therefore demands preformed primer-template junction with free 3' OH precisely positioned in active site opposite template. In cells primase, specialized RNA polymerase, synthesizes short RNA primers complementary to template providing required hydroxyl. This obligate primer dependence ensures replication starts only at regulated origins and prevents random DNA synthesis elsewhere.

Ref: Berg et al., Biochemistry, 9th ed., Chapter 27: Requirement of primer 3'OH for DNA polymerase activity

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Replication Basics

PCNA in eukaryotes acts as

Eukaryotic replicative polymerases delta and epsilon are intrinsically poorly processive adding only tens of nucleotides per binding. Processivity depends on circular trimeric protein PCNA, proliferating cell nuclear antigen, that completely encircles duplex DNA forming sliding clamp. Loaded by RFC clamp loader using ATP hydrolysis, PCNA slides freely and tethers polymerase via PIP-box interaction preventing dissociation. It dramatically increases synthesis from tens to thousands of nucleotides processively. PCNA also coordinates Okazaki maturation, mismatch repair, translesion synthesis serving as central replication hub analogous to bacterial beta clamp dimer.

Ref: Lodish et al., Molecular Cell Biology, 9th ed., Chapter 11: PCNA as eukaryotic sliding clamp and processivity factor

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Replication Basics

Okazaki fragments are formed on

Okazaki fragments, 1000 to 2000 nucleotides long in bacteria and 100 to 250 in eukaryotes, are short nascent DNA segments observed during brief pulse labeling experiments. They appear exclusively on lagging strand because its template synthesized opposite to fork movement. Each fragment begins with 10 to 12 nucleotide RNA primer made by primase that provides essential 3' OH for Pol III extension. Polymerase extends until reaching previous fragment then displaces or awaits processing. Leading strand shows no such fragments under normal physiological conditions.

Ref: Alberts et al., Molecular Biology of the Cell, 7th ed., Chapter 5: Okazaki fragments on lagging strand

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Replication Basics

Lagging strand synthesis is

Both template strands are antiparallel, but replication fork moves unidirectionally. Leading strand polymerase follows helicase smoothly synthesizing continuously. Lagging strand template runs 5' to 3' toward fork, requiring polymerase to synthesize away from fork advancement. As helicase unwinds parental duplex, new single-stranded DNA exposed discontinuously behind fork. Primase synthesizes short RNA primers at intervals, Pol III extends each into Okazaki fragment that later becomes processed and joined. This semi-discontinuous mode elegantly solves antiparallel constraint without violating fundamental 5' to 3' polymerization chemistry.

Ref: NCBI Bookshelf, Molecular Cell Biology, Lodish, Figure 11-21: Discontinuous synthesis of lagging strand

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Replication Basics

Leading strand synthesis requires primers

At replication fork, leading strand synthesis runs continuously toward fork movement with same polarity as unwinding. Because duplex opens progressively ahead, polymerase can track helicase without interruption. DNA polymerase requires pre-existing 3' OH for extension, supplied by primase only once at origin. Single RNA primer about 10 nucleotides synthesized by DnaG provides initial hydroxyl for Pol III, thereafter Pol III extends uninterrupted for hundreds of kilobases. Lagging strand oriented opposite cannot do this and therefore needs repeated priming generating Okazaki fragments.

Ref: Alberts et al., Molecular Biology of the Cell, 7th ed., Chapter 5: Leading strand continuous synthesis and priming

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Replication Basics

Proofreading occurs at which end of growing strand?

DNA polymerases universally synthesize only 5' to 3' direction adding nucleotides to free 3' hydroxyl of growing primer chain. Proofreading exonuclease activity scans most recently added nucleotide because misincorporation creates mismatch at growing tip distorting duplex geometry and slowing next polymerization step dramatically. Polymerase then reverses one step moving branched 3' terminus from polymerase site to distinct exonuclease site that specifically hydrolyzes phosphodiester bond from 3' end preferentially. Excision cannot occur internally or at 5' terminus restricting surveillance efficiently to active 3' terminus where errors arise during elongation.

Ref: Lodish et al., Molecular Cell Biology, 9th ed., Chapter 11: Proofreading at the 3' terminus during replication

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Replication Basics

Tautomeric shift in guanine can cause pairing with

Purines and pyrimidines normally exist in keto-amino tautomers that form canonical Watson-Crick pairs G-C and A-T. Rare enol-imino tautomers relocate hydrogen bonding donors and acceptors altering pairing face. A transient tautomeric shift converts guanine to enol form presenting hydrogen bonding pattern similar to adenine, allowing stable pairing with thymine instead of cytosine. If uncorrected before next round of replication, replication past this mispair fixes G-C to A-T transition mutation. Phenomenon underlies spontaneous point mutations and explains mutagenesis by base analogs like bromouracil.

Ref: Watson JD et al., Molecular Biology of the Gene, 7th ed., Chapter 9: Rare tautomeric shifts and mispairing

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Replication Basics

Error rate of DNA replication in E. coli is approximately

Overall replication fidelity in E. coli reflects three sequential filters acting in series. Base selectivity by Pol III active site contributes roughly 10^-5 error rate, proofreading by epsilon subunit adds 10^-2 improvement, and methyl-directed mismatch repair provides another 100 to 1000-fold correction. Combined outcome reaches one mistake per 10^9 to 10^10 nucleotides incorporated, equivalent to less than one error per genome duplication of 4.6 megabases. This extreme accuracy preserves genome stability despite rapid polymerization near 1000 nucleotides per second at each replication fork.

Ref: Alberts et al., Molecular Biology of the Cell, 7th ed., Chapter 5: Fidelity of DNA replication and mutation rates

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Replication Basics

rNTPs are incorporated less frequently because

Cellular rNTP concentrations exceed dNTPs roughly tenfold, yet DNA polymerases insert ribonucleotides rarely, about once per thousand nucleotides. Selectivity depends on steric gate residue in active site. In E. coli Pol I this is Glu-710 backed by Phe-762 creating clash with 2' hydroxyl of ribose sugar. Family B and Y polymerases use tyrosine or phenylalanine similarly blocking ribose. The clash prevents proper alignment of alpha-phosphate, raising energetic barrier for catalysis. Mutating gate dramatically increases rNMP incorporation causing alkali-sensitive strand breaks threatening genome stability.

Ref: NCBI Bookshelf, Biochemistry: Berg et al., Section on steric gate preventing rNTP incorporation by DNA polymerases

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Replication Basics

Incorrect nucleotide incorporation is reduced mainly by

Even highly selective polymerases occasionally incorporate non-complementary nucleotides at rates near 10^-5 during rapid synthesis. Immediate correction relies heavily on intrinsic 3' to 5' exonuclease proofreading activity located in separate domain. When misincorporation distorts primer-template duplex geometry, polymerase stalls and transfers frayed 3' end from polymerase active site to exonuclease site that hydrolyzes phosphodiester bond and excises incorrect base. Strand then repositions into polymerase site for continued accurate synthesis. This kinetic proofreading step improves fidelity about 100-fold reducing errors to 10^-7 before mismatch repair.

Ref: Lodish et al., Molecular Cell Biology, 9th ed., Chapter 11: DNA replication fidelity and proofreading exonuclease

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Replication Basics

Metal ion required for DNA polymerase activity is

DNA synthesis proceeds via nucleophilic attack of primer 3' hydroxyl on alpha-phosphate of incoming dNTP. Two Mg2+ ions, coordinated by conserved aspartate residues in polymerase active site, orchestrate catalysis. One ion activates 3'OH lowering pKa for attack, the other stabilizes pentacovalent transition state and facilitates pyrophosphate departure. Without Mg2+, phosphodiester bond formation stalls entirely. This two-metal mechanism explains why chelators like EDTA inhibit polymerization, why Ca2+ fails to substitute effectively, and why high fidelity polymerases absolutely require magnesium for both polymerase and exonuclease activities.

Ref: Alberts et al., Molecular Biology of the Cell, 7th ed., Chapter 5: DNA Replication Mechanisms, Two-metal-ion catalysis

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