Replication in Prokaryotes

Replication in Prokaryotes

Joining of Okazaki fragments requires

Okazaki fragment synthesis inevitably leaves gaps between fragments after primer removal. Pol I synthesizes DNA to fill those short single-strand gaps using upstream 3' OH as primer ensuring precise replacement of RNA with DNA in 5' to 3' direction. Once filling complete, adjacent fragments remain connected by single phosphodiester nick because polymerase cannot create final bond between 3' hydroxyl and 5' phosphate. DNA ligase using NAD+ cofactor in bacteria hydrolyzes to close nick forming continuous phosphodiester backbone. Requirement for both enzymes couples gap filling and final ligation.

Ref: Alberts et al., Molecular Biology of the Cell, 7th ed., Chapter 5: Joining Okazaki fragments by Pol I and DNA ligase

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Replication in Prokaryotes

Nick translation is performed by

Nick translation is coupled exonuclease and polymerase reaction moving nick position along duplex without net synthesis. Pol I binds nick bearing free 3' OH, degrades downstream strand via 5' to 3' exonuclease while extending 3' end with polymerase activity incorporating new dNTPs, effectively translating nick along DNA. Property historically exploited to label DNA probes with radioactive dNTPs replacing strand segment uniformly. Pol III cannot degrade downstream duplex, ligase only seals nicks without synthesis. In vivo role of Pol I during Okazaki fragment processing directly mirrors laboratory nick translation mechanism.

Ref: Berg et al., Biochemistry, 9th ed., Chapter 27: Nick translation mechanism by DNA polymerase I

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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

Decatenation of daughter chromosomes is done by

After circular chromosome duplication, two daughter molecules become interlinked as catenanes resembling linked rings generated naturally during replication termination and intertwining. Topoisomerase IV, type II topoisomerase of ParC and ParE subunits as C2E2 tetramer, performs decatenation by introducing transient double-strand breaks in one duplex, passing other duplex through opening, and resealing using ATP hydrolysis energy. DNA gyrase primarily introduces supercoils, topoisomerase I relaxes negative supercoils but cannot decatenate due to single-strand mechanism. Topo IV activity essential for chromosome segregation before cell division.

Ref: Alberts et al., Molecular Biology of the Cell, 7th ed., Chapter 5: Topoisomerase IV decatenation of sister chromosomes after replication

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Replication in Prokaryotes

Ter sites are recognized by

Ter sites are specific DNA sequences within termination region where replication fork arrest occurs. Tus, terminus utilization substance, 36 kilodalton monomer, recognizes 23 base pair ter consensus through major groove contacts forming tight stoichiometric complex with Kd near one nanomolar. Upon approach of DnaB helicase from blocking direction, C-terminal region of Tus locks onto helicase halting advancement. DnaA and SeqA proteins bind origin not terminus sequence. Tus-ter pair thus functions as replication fork trap ensuring forks meet within termination zone avoiding over-replication.

Ref: NCBI Bookshelf, Lewin Genes: Tus protein recognizes ter sites forming polar fork trap, replication termination

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Replication in Prokaryotes

Termination of replication involves binding of Tus protein to

E. coli chromosome contains terminus region opposite oriC harboring ten ter sites each 23 base pairs oriented to trap forks arriving from either direction. Tus protein binds ter as monomer and when replication fork helicase DnaB encounters Tus-ter complex in non-permissive orientation, Tus interacts with DnaB blocking unwinding and arresting fork progression. This polar barrier prevents over-replication beyond terminus and ensures fork fusion within terminus region. OriC functions in initiation, DUE unwinds initially, but Tus-ter provides authentic termination checkpoint enforcing completion.

Ref: Lodish et al., Molecular Cell Biology, 9th ed., Chapter 11: Tus binding to ter sites terminates replication fork progression

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Replication in Prokaryotes

DNA gyrase is a type of

Topoisomerases classified mechanistically by whether cleavage of one versus two DNA strands occurs during catalytic cycle. Type I cuts single strand allowing rotation to change linking number in steps of one. Type II cuts both strands, passes intact duplex through break, alters linking number by two. DNA gyrase and topoisomerase IV are bacterial type II enzymes requiring ATP and Mg2+ forming transient covalent phosphotyrosine intermediates. Gyrase uniquely introduces negative supercoils and removes positive supercoils ahead of replication, while topo IV primarily decatenates chromosomes. Double-strand mechanism distinguishes gyrase from type I enzymes.

Ref: Berg et al., Biochemistry, 9th ed., Chapter 27: Type II topoisomerases, DNA gyrase mechanism and classification

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Replication in Prokaryotes

Protein that relieves positive supercoiling is

Unwinding helicase generates positive supercoils ahead of fork increasing helical twist and resisting further strand separation requiring topoisomerase relief. DNA gyrase, type II topoisomerase composed of GyrA and GyrB subunits as A2B2 tetramer, introduces negative supercoils using ATP hydrolysis to counterbalance positive supercoiling essentially removing overwinding stress. It makes transient double-strand cuts, passes another segment through break, and religates. SSB binds single strands but does not resolve topology, ligase seals nicks only. Gyrase thus prevents fork stall and facilitates rapid fork progression.

Ref: Alberts et al., Molecular Biology of the Cell, 7th ed., Chapter 5: DNA gyrase relieving positive supercoils ahead of fork

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Replication in Prokaryotes

Replication in E. coli is

Initiation at oriC recruits two DnaB helicases loading onto opposite parental strands, each advancing outward in opposite directions from origin. Replication then proceeds bidirectionally with two forks moving away from origin toward terminus region opposite chromosome. Bidirectional synthesis doubles overall speed of genome duplication and is typical of bacterial archaeal and eukaryotic chromosomes. Unidirectional replication occurs in some plasmids via rolling circle mechanism but not chromosome. Bidirectionality explains bubble structures observed in electron micrographs and timing of genetic marker replication around circular map.

Ref: Lodish et al., Molecular Cell Biology, 9th ed., Chapter 11: Bidirectional replication from oriC origin

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Replication in Prokaryotes

Replication in E. coli proceeds by

Chromosome replication in E. coli visualized historically by autoradiography resembles Greek letter theta, where central replication bubble expands between two diverging forks within circular duplex. As synthesis proceeds, unreplicated parental segment plus two replicated arms resemble theta shape. This contrasting model contrasts with sigma rolling circle mode used by many plasmids and phage lambda late replication where one end spills out as concatamer, and linear mode of eukaryotes with multiple origins. Theta mode preserves circular integrity and permits efficient bidirectional progression around circle.

Ref: Alberts et al., Molecular Biology of the Cell, 7th ed., Chapter 5: Theta mode replication of circular bacterial chromosome

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Replication in Prokaryotes

Error-prone polymerases in E. coli are

E. coli maintains three error-prone polymerases under SOS control: Pol II encoded by polB for restart after damage, Pol IV encoded by dinB, and Pol V encoded by umuC-umuD for translesion synthesis past lesions. These belong to B and Y families lacking intrinsic 3' to 5' proofreading exonuclease and possessing spacious active sites tolerant of bulky lesions but misinserting bases frequently. Pol I and Pol III are high-fidelity replicative enzymes with strong proofreading exonuclease not classified error-prone. Deletion of dinB and umuC reduces UV-induced mutagenesis drastically but increases sensitivity.

Ref: NCBI Bookshelf, SOS polymerases overview: Pol II, IV, V as error-prone translesion polymerases versus Pol I, III

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Replication in Prokaryotes

DNA polymerase V is part of

Severe DNA damage causes accumulation of single-stranded DNA activating RecA to stimulate autocleavage of LexA repressor derepressing over 40 SOS genes regulon. Among strongly induced are umuD and umuC genes encoding Pol V, Y-family error-prone polymerase capable of copying past pyrimidine dimers that block Pol III. Pol V requires RecA nucleoprotein filament for activation as mutasome complex. Its expression relatively late in SOS response provides last-resort translesion synthesis when excision repair incomplete enabling survival despite persisting lesions but dramatically increasing mutation rate.

Ref: Lewin Genes XII, Chapter 16: SOS response induction of Pol V UmuC-D for translesion synthesis

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