#exonuclease

Replication in Prokaryotes

RNA primer removal in prokaryotes is done by

Lagging strand synthesis leaves behind RNA primers that must be removed to create contiguous DNA strand before ligation. In prokaryotes DNA polymerase I performs this task using intrinsic 5' to 3' exonuclease that degrades RNA while simultaneously polymerizing DNA forward process called nick translation efficiently replacing RNA with DNA. RNase H also can clip RNA primers but main removal during replication is Pol I specifically. Pol III lacks this forward exonuclease, Pol II has none. After Pol I fills gap, remaining nick sealed by NAD-dependent DNA ligase.

Ref: Lodish et al., Molecular Cell Biology, 9th ed., Chapter 11: Pol I 5' to 3' exonuclease removes RNA primers in Okazaki processing

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Replication in Prokaryotes

Proofreading activity of Pol III is due to

High-fidelity replication by Pol III relies critically on epsilon subunit, potent 3' to 5' exonuclease belonging to DnaQ family. When alpha polymerase misincorporates base causing mismatch and frayed terminus, primer moves from polymerase site to epsilon active site where phosphodiester bond hydrolyzed releasing monophosphate. Epsilon requires two Mg2+ ions and prefers single-stranded mismatched ends over duplex. Mutator dnaQ mutants lacking epsilon exhibit 1000-fold increased spontaneous mutation rates illustrating proofreading contribution. Theta subunit wraps around epsilon protecting from heat denaturation enhancing activity.

Ref: Lodish et al., Molecular Cell Biology, 9th ed., Chapter 11: Epsilon subunit proofreading in Pol III fidelity

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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

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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