DNA Replication Fidelity and Initiation: Comprehensive Notes
Mistakes in DNA replication: possible errors and why they matter
Three obvious potential errors when the DNA is being replicated (not an exhaustive list, but the key ones highlighted):
Incorrect nucleotide incorporation: while the chain is growing, the wrong nucleotide may be added (e.g., a
T should be paired but a G is inserted). This is an immediate mutation if it occurs.Accidental RNA nucleotides in DNA: RNA nucleotides could theoretically be incorporated despite RNA primers being present; the question is raised whether RNA bases might be inserted as incorrect substrates.
Backbone nicks: if the phosphate–sugar backbone has nicks, the DNA molecule can break into fragments during replication, preventing proper template alignment. There must be no nicks for faithful replication.
How polymerase recognizes and handles incorrect base pairs at the active site: the DNA polymerase must recognize all four correct base-pairs yet reject incorrect ones.
The active site must accommodate all four correct base-pairs but reject incorrect combinations; this is a challenge because the enzyme handles multiple possibilities.
As an enzyme, polymerase exhibits typical features: shape-based discrimination, binding affinity, and an induced-fit mechanism.
The active-site region (including a key helically positioned segment) moves to enclose the incoming nucleotide only if it fits correctly; this is the induced-fit step.
Fidelity checks at the active site (three sequential checks):
Shape compatibility: the overall shape, size, and width of a base-pair are important; even if hydrogen bonds could form, a misfit will be ejected due to steric clashes with the active site edges.
Binding affinity via minor-groove interactions: two amino-acid side chains in the polymerase interact with the base-pair in the minor groove to check for a correct base-pair geometry. These two contacts work for all four correct base-pair combinations (A·T and G·C, in either orientation); if these contacts are absent, the base-pair is not held in place and may be released.
Induced-fit closing: the enzyme only adopts the final active conformation if the base-pair fits correctly; the key helix (in the active site) moves to enclose the nucleotide if correct; otherwise, the nucleotide is not properly enclosed and no bond is formed.
The role of magnesium and the catalytic setup:
The active process requires Mg^{2+} ions coordinated by aspartate residues within the active site to hold substrates in place and promote bond formation.
The incoming triphosphate nucleotide is escorted into the active site, where it can form phosphodiester bonds only when correctly paired.
Proofreading reduces the error rate dramatically (from 10^{-5}):
Without proofreading, the error rate is about:
$10^{-5}$ per base incorporated (approximately 1 error per 100{,}000 nucleotides).
With proofreading by the 3'→5' exonuclease activity of DNA polymerase, the error rate drops by roughly a factor of 100, to about:
$10^{-7}$ per base, i.e., about 1 error per 10{,}000{,}000 nucleotides.
Consequence of the raw error rate (for a model bacterium): about 46 point mutations per replication would be produced, which is not sustainable.
How proofreading works (3'→5' exonuclease):
The polymerase has a secondary active site dedicated to exonuclease activity that runs 3'→5'.
If the last incorporated nucleotide is incorrect, the end of the growing DNA strand is repositioned to the exonuclease site and the incorrect nucleotide is removed, then synthesis resumes.
Mechanistically, when a correct base is in place and the helix is properly closed, proofreading is not engaged; when an incorrectly paired nucleotide is present, the end reverses into the exonuclease site and is cleaved.
The structural model of DNA polymerase is often described as a hand: fingers, palm, and thumb (with the exonuclease active site located in the wrist region). The palm is where DNA synthesis occurs; the fingers move to guide the nucleotide; the wrist positions the 3' end for possible exonuclease editing.
If a mismatch slows the polymerase (due to incorrect base-pairing geometry), the enzyme effectively pauses, giving the exonuclease time to remove the last nucleotide and restore a correct terminus.
Roles of polymerase I and polymerase III in proofreading and repair:
Polymerase III: primary replicative polymerase; responsible for synthesis and the 3'→5' proofreading exonuclease activity (the subtraction of incorrect nucleotides at the end of the growing strand).
Polymerase I: additional proofreading capability, with both a 3'→5' exonuclease (like polymerase III) and a 5'→3' exonuclease that can remove nucleotides ahead of the growing chain.
Pol I can recognize and bind to nicks and proceed to replace nucleotides (forward repair) by moving ahead of the nick and replacing nucleotides, handling non-correct nucleotides as well as correct ones.
Experimental note: after treating with a protease, polymerase I can be separated into an N-terminal fragment containing the 5'→3' exonuclease and a C-terminal fragment containing polymerase plus 3'→5' exonuclease; this allows purification of the basic polymerase with proofreading capability for lab use.
This arrangement forms a robust, multi-layered proofreading system to reduce errors beyond what polymerase III alone could achieve.
Okazaki fragments and nick repair in replication:
The lagging strand is synthesized discontinuously in short fragments (Okazaki fragments) that require processing and ligation.
DNA ligase seals nicks between fragments to create a continuous strand.
If a mutant DNA ligase cannot seal nicks, leading and lagging strands show increased fragmentation, revealing the dependence on ligation for continuous DNA synthesis.
There are pseudo-Okazaki fragments on the leading strand as well during normal replication due to processing events; the end result is a need to seal nicks on both strands to achieve two intact chromosomes.
Nicks can arise naturally during replication (e.g., during primer processing) and must be sealed promptly to avoid fragmentation.
RNA primers and the avoidance of RNA in DNA:
RNA primers are required to start synthesis; primase (an RNA polymerase) generates short RNA primers (approximately 10–18 nucleotides) at the replication fork.
Primase activity is enhanced when bound to the helicase; the presence of helicase increases primase activity via cooperative interactions.
The primer ends provide a 3'-OH for DNA polymerase III to begin elongation; primers are later removed and replaced with DNA.
RNA nucleotides are generally not incorporated into DNA by DNA polymerases due to substrate discrimination: the ribose sugar (with an extra hydroxyl) makes RNA nucleotides unsuitable substrates for DNA synthesis, so RNA is not used as part of the final DNA chain (beyond the primers).
Primers themselves are removed later by polymerase I activity during Okazaki fragment processing.
RNA in DNA and the problem of uracil (U) in DNA:
In replication, the presence of U is not immediately problematic if it is part of a primer or occasional misincorporation that is corrected, but U should not persist in DNA because it can lead to mutations.
A common source of U in DNA is deamination of cytosine (C → U). This can cause a GC pair to deaminate into a GU mispair, which can be transcribed into an AU pair on replication, creating a mutation.
Another source of U is misincorporation when dUTP is used as a substrate instead of dTTP during replication.
U in DNA is actively detected and removed by the Ung pathway:
Uracil-DNA glycosylase (UNG) recognizes and removes uracil bases from DNA, leaving an abasic site (AP site).
AP endonuclease recognizes abasic sites and cleaves the phosphodiester backbone to create a nick, generating an abasic nick (AP site nick).
After ung mutants (lacking UNG) are present, uracil cannot be removed, so uracils accumulate and lead to fragmentation when the nick is processed; in ung mutants, nicks and fragments are not generated by this pathway.
Polymerase I can recognize these nicks anywhere and proceed to remove the nucleotides ahead of the nick, replacing with correct ones, completing the repair process.
In normal cells, after UNG removes a uracil and an AP endonuclease makes a nick, polymerase I fills in the gap and ligase seals the nick to restore intact DNA.
Consequences of dUTP presence and U incorporation in DNA: experimental evidence
The pathway that converts UMP to dUTP can lead to U incorporation with a typical ratio of about 1 U per 300 T's, which means that at a given site there is a non-negligible chance of inserting U before proofreading and repair catch up.
Since there are four possible nucleotides, a rough estimate yields about one U per ~1200 nucleotides, when accounting for insertion probabilities and base composition, leading to thousands of U residues across a large genome if unrepaired.
If a polymerase mutation (enzyme 4 in the described context) blocks the conversion of dUTP to dTTP, more U gets incorporated, resulting in shorter fragments being observed due to more frequent uracil incorporation and removal events.
U is considered mutagenic because deamination of C to U changes the information content; after replication, a C that deaminates to U can lead to a GC pair becoming AT (AU in the replicated strand), a fixed mutation if not repaired.
Therefore, the presence of U is treated as evidence of potential DNA damage and must be repaired to prevent mutation.
Deamination damage and mutation consequences (C → U): a concrete example
Start with a GC pair; during replication, if cytosine is deaminated to U on one strand, the two strands separate and are replicated.
The original C that became U now pairs with A (since U pairs with A) during replication, yielding an AU pair instead of GC on the new strands.
This “locked-in” mutation cannot be corrected by standard proofreading, since the mismatched base now exists as U in the template; hence repair pathways must remove the U to prevent mutation.
The presence of U can thus be mutagenic regardless of whether it originated from replication misincorporation or deamination damage.
Initiation of replication at origin (oriC) in E. coli: an overview
Every circular piece of DNA has an origin of replication (ori); in E. coli, oriC (RDC in the transcript) is a defined region about 245 base pairs long, AT-rich (weaker base pairs) to facilitate strand separation.
The oriC region contains modular repeats: described as four nine-base-pair repeats and three 13-base-pair repeats in the transcript (note: these numbers reflect the specific example in the lecture transcript).
Step 1: DNA A protein (gene product) binds to the 9-bp repeats in oriC.
The binding by DNA A, and the subsequent wrapping and twisting of DNA around the protein, creates torsional strain that causes the 13-bp “beads” to pop open, forming a bubble and exposing single-stranded DNA.
Step 2: This open region becomes a binding site for the helicase loader complex: DNA C binds to DNA B (the helicase) and together they move toward the three-prime ends of the strands.
Step 3: After an initial unwinding, when about 65 nucleotides have been unwound (single-stranded region of ~65 nt), additional helicase loading occurs and primase can bind, forming the replication fork priming site.
Primase, primers, and the start of synthesis at the fork
Primase is an RNA polymerase (distinct from the RNA polymerase used in transcription) that binds to single-stranded DNA at the fork and synthesizes an RNA primer complementary to the exposed DNA.
The primer is typically about 10 nucleotides long; its function is to provide a starting 3'-OH for DNA polymerase III to begin synthesis.
Primase activity is enhanced when bound to the helicase; more primase binding leads to more primer synthesis (cooperative binding).
The primer is synthesized in the 5'→3' direction, and the primer ends serve as initiation points for DNA synthesis.
A replication fork protection mechanism exists: single-stranded DNA binding proteins (SSB) coat exposed single strands to prevent re-annealing and to keep the fork open for synthesis.
Single-stranded DNA binding protein (SSB) and primer clearance
SSB is a tetrameric protein that coats exposed ssDNA, preventing re-annealing and protecting the DNA.
Binding is cooperative: when one SSB binds, additional SSBs rapidly accumulate to cover the exposed single-stranded region.
The end of the primer that is approached by polymerase III must be accessible; SSB needs to be displaced as polymerase advances.
SSB binding does not mask primer bases that could be recognized by sequence-specific proteins; it mainly protects ssDNA and maintains fork structure.
DNA polymerase III recognition and initiation of elongation
DNA polymerase III holoenzyme recognizes the end of the RNA primer and the presence of SSB at the fork to bind and initiate DNA synthesis.
Once bound, Polymerase III begins elongation along the new strand, extending the DNA in the 5'→3' direction.
The combination of primer end recognition and SSB presence helps ensure efficient, accurate initiation of elongation at the replication fork.
Three stages of macromolecule replication
Initiation: origin recognition (e.g., oriC in E. coli), origin opening, helicase loading, primer synthesis initiation.
Elongation: synthesis of new DNA by DNA polymerases with proofreading and repair systems in place.
Termination: processes to end replication and ensure two complete, intact chromosomes are produced.
Some practical connections and implications
The proofreading and repair pathways (Pol III, Pol I, Ung, AP endonuclease, ligase) work together to maintain genome integrity during rapid replication.
Failures in these pathways (e.g., ligase mutants, ung mutants, or polymerase mutations) lead to characteristic fragment patterns and increased mutation rates, which are often used in experimental genetics to infer function.
The replication machinery illustrates an integrated system where initiation, elongation, and termination are tightly coordinated with repair and damage-control pathways to preserve genetic information across generations.
Key concepts and terms to remember (summary):
Base-pair fidelity checks: shape fit, minor-groove binding (two amino acids interacting with base pairs across all four correct combinations), induced-fit closing, and Mg^{2+}-coordinated catalysis.
Error rate without proofreading: $10^{-5}$; with proofreading: around $10^{-7}$.
3'→5' exonuclease (Pol III) and 5'→3' exonuclease (Pol I) activities; structural hand model (fingers, palm, thumb; exonuclease at the wrist).
Okazaki fragments on the lagging strand; role of ligase in sealing nicks; pseudo-Okazaki fragments on the leading strand when ligase is compromised.
Uracil in DNA (U): sources include dUTP incorporation and deamination of C to U; UNG removes U to create an abasic site; AP endonuclease creates a nick; Pol I fills in and ligase seals; ung mutants accumulate U and result in different fragment patterns.
dUTP/dTTP balance and U incorporation frequency: roughly 1 U per 300 T's; about one U per ~1200 nucleotides overall; U is mutagenic if not repaired.
oriC structure in E. coli: AT-rich region, ~245 bp, repeats with DNA A binding; bubble formation and helicase loading by DNA B/DNA C; primase primer synthesis; SSB protection; initiation of elongation by Pol III.
Title
DNA Replication Fidelity, Proofreading, and Initiation (Comprehensive Notes)