Prokaryotic DNA replication is anabolic process by which prokaryotes duplicate their genomic DNA into another copy, which is later transferred to daughter bacterial cell. DNA replication in prokaryotes especially in E. coli (model microorganism) was well studied.
DNA replication in prokaryotes (e.g. E. coli)
DNA replication is not random process, so replication start at particular site in chromosome.
The genomic DNA of prokaryotes is organized into a single replicon. Replicon is the unit of replication, that contains 4.6 Mbp size and it indicates an entire DNA that can replicate as a single unit.
Each replicon contains a single origin called as chromosomal origin or ori-C. Ori-C is 244 base pair sequence that contains different types of DNA replicative sequences. The unit (replicon) of DNA replicated from origin site by biderectionally or unidirectionally to terminus site. The total genome of E. coli is only one replicon size (approximately 4.6 Mbp), hence whole genome in bacteria replicates as a single unit.
Proteins required for prokaryotic DNA replication
DNA replication is a complex process accrued in three steps initiation, elongation and termination. In these processes involve various types of enzymes and proteins. More than 20 different proteins play a role in prokaryotic DNA replication. These proteins have been classified on the bases of their biochemical functions in different strategies of prokaryotic DNA replication.
1) Proteins required for replication initiation:
DnaA (initiator), Single strand binding protein (SSB), DnaB (helicase), DnaC (helicase loader) and DnaG (primase).
2) Proteins involved in DNA chain elongation and ligation of Okazaki fragments:
DNA polymerase (adds the nucleotides) , SSB (stabilizes the single strands), DNA gyrase (unwinds the strands) and DNA ligase (joins the Okazaki fragments).
3) Proteins required for replication termination and separation of daughter DNA molecules
Tus protein (Terminus binding protein) and DNA topoisomerase (separates daughter molecules)
The major repetitive sequences present in ori-c are……
1) Four 9 mer sequences:- These are the binding sites for the replication initiation protein such as Dna-A.
2) 3.13 mer sequences:- These are AT-rich sequences and undergo initial melting during the replication initiation.
3) Several GATC sequences:- These sequences are palindromes and read the same both the strands. The adenine base in the GATC sequence can be methylated and methylation status determines the occurrence of replication initiation. if GATC methylated in both the strands then the initiation protein can bind to the DNA and replication can be initiated. whereas if GATC is hemimethylated then the initiation protein cannot bind to the DNA thereby replication cannot be initiated.
Mechanism of DNA replication in prokaryotes in several steps
Step 1: The replication initiation begins with the binding of Dna-A to five the 9 mer sequences present in the oriC region. The binding of Dna-A to 9 mer sequence will be initially very week and will have low binding efficiency. However, binding of first Dna-A protein exhibits cooperativity, it resulted into increasing the binding efficiency of several Dna-A proteins to the 9mere sequences.
Step 2. The binding of Dna-A followed by a binding of Histone-like protein called Hu proteins. The binding of Dna-A and Hu protein at 9 mer sequence cause a conformational change in adjacent 13 mere sequence leading to the localized melting of DNA or strand separation. This process forms open complex and requires ATP.
Step 3. The localized unwinding of DNA in the 13 mer sequence promotes the binding of helix destabilization proteins SSBs (single strand binding proteins).
Step 4. DNA helicase Dna-B (hexamere), is loaded on to the 13 mere sequence by the helicase loader (Dna-C). Hexameric identical subunits of DNA helicase clamps around each of the two single strands formed as open complex between DnaA and oriC. This process require ATP. As the unwinding of DNA occurs in the presence of DNA helicase, the initiation protein DnaA and Hu proteins are gradually removed from 9 mer sequences.
Step 5. DNA polymerase can only elongate existing primer strands of DNA or RNA. Hence. unwinding of DNA by DNA helicase facilitate the primase enzyme (Dna-G) binds to the helicase and forms the primosome complex. DNA primase synthesizes the first primer for the leading strand.
Step 6: The formation of the primer promotes the loading of the sliding clamp by the clamp loader (γ-complex). The core DNA polymerase is then added to the sliding clamp. Once the core polymerase is loaded then it begins the extension of the primer.
Step 7: When primer of leading strand is extended by few nucleotides then the first primer for the lagging strand is synthesized. The primer synthesis activity will have to be repeated several times during the formation of lagging strand, which is a synthesis in the form of Okazaki fragments, ranging in size from 1000 nucleotides to 2000 nucleotides.
In order to facilitate the same rate of replication in both leading and lagging strand, the lagging strand template makes loop formation, so that both the strands will be replicated at the same duration. The usual rate of replication by DNA polymerase-III is 200 to 1000 nucleotides incorporated per second, under optimal conditions DNA polymerase can add 15000 nucleotides per minute.
Step 8: The elongation phase continues until the polymerases reach the termination sequence, each DNA strand contains a Ter sequence opposite to origin sequence, the two Ter sequences in E. coli are Ter EDA and Ter BCF. Ter sequence is a binding site for Tus sequence
Step 9: When the Tus protein binds to the Ter sequence then it will not allow the DNA polymerase to move at the Ter region and therefore replication will be terminated.
Step 10: In the case of the lagging strand, the Okazaki fragments can join together by DNA ligase to complete the replication process.
- The time required for replication of whole genome and doubling time in E. coli is virtually constant at 37ºC.
- The time required for the replication of chromosome is 40 min and 20 min for doubling the cell.
- To continue the doubling of cells, the organism should develop the capacity to initiate the second round of replication before the previous round has terminated.
- Because of this advanced initiation process, on a single chromosome as many as six replication forks may be active, this process called as Dichotomous replication.