Prokaryotic DNA Replication

Prokaryotic DNA Replication

DNA Replication in Prokaryotes is the process by which a prokaryotic genetic material (DNA) is copied and transmitted to the daughter cells.

Structure of DNA

DNA (deoxyribonucleic acid) is a polymer of deoxyribo nucleotide. Each deoxyribo nucleotide molecule is composed of 3 groups.

  • Deoxyribose sugar molecule
  • Nitrogen base
  • Phosphate group

Deoxyribose is a cyclic, five carbon sugar molecule.

Prokaryotic DNA Replication
Prokaryotic DNA Replication

There are two types of Nitrogen bases: Purin (2 rings – Adenine andGuanine) and Pyrimidine (1 ring – Thymine and Cytosine). The base is attached to the 5’C sugar molecule. It’s covalently bond by glycosidic bond. 5’C molecule can be covalently bonded with one or more phosphate groups of DNA form what is often called a back bone of the DNA molecule. The nucleotide without the phosphate group is known as a nucleoside.

DNA Chain

The phosphate group attached to the 5’C of the deoxyribose sugar molecule of one nucleotide is attached to the 3’C of the next nucleotide. The bond is called the phosphodiester bond. The nucleotides found at the ends of the linear piece of DNA have properties that are biochemically important in orienting the DNA strand. Each DNA strand has what is called as 5’end and 3’end. 5’ end of the DNA strand will always have a phosphate group attached to the 5’C of it’s terminal nucleotide. The other end of the strand is called 3’ end. It will always have a hydroxyl group attached to the 3’C.

The nucleotide sequence is a sequence of bases found in the DNA molecule. Usually it is written from 5’-3’. DNA polymer commonly occur as a double strand helical molecule. The two separate nucleotide chains are held together by forming H bonds between bases. Adenines pair only with Thymine forming two H bonds. Guanine pairs only with Cytosine forming three H bonds. So, each Adenine and Thymine pair and each Guanine and Cytosine pair in DNA is called complementary base pairs.

Most of the bacterial chromosomes are circular in nature. They are covalently circular. These covalently closed circules can be twisted which is called super coiling. In general, bacterial DNA are negatively super coil (anti-clockwise). Super coiling introduces further stress in to DNA. So, that further twisting can occur forming loops. The super coil DNA is a compact mass of DNA.

Antiparallel construction

Two nucleotide chains running opposite orientation in the double helix. That is one strand runs from 5’ to 3’ while its complementary strand runs antiparallelly 3’ to 5’ direction. Two strands of DNA are rap around to form the double helix of DNA. Therefore, the helix have two grooves between two strands; Major groove (2.2nm) and Minor groove (1.2nm). One helical turn is about to base pairs.

Structure of RNA

RNA (Ribonucleic acid) is a polymer of ribonucleotide. Each ribonucleotide is composed of three groups as ribose sugar molecule, nitrogenous base and phosphate group.

Unlike in DNA, RNA does not have Thymine. Instead of Thymine, RNA has Uracil. RNA is a single standard nucleic acid. The base is attached to the 5’C in the sugar molecule. It’s covalently bonded through glycosidic bond. RNA strand is also contains 5’end and with the phosphate group and the 3’ end is terminal end with the hydroxyl group.

Prokaryotic DNA Replication

DNA Replication in Prokaryotes

In the late 1950s, 3 different mechanisms were proposed for the explain DNA Replication in Prokaryotes.

  • Conservative model – Both parental strands stay together.
  • Semi conservation – The double-stranded DNA contains one parental and one daughter strand.
  • Parental and daughter DNA are interspersed in both strands.

Basic process of replication involves polymerizing or linking nucleotides of DNA in to long chains using the sequence of another strand as guide. The guiding strand is called the parental strand. So, it acts as the template for the synthesis of the new daughter strand. When the replication process parental double helix unwinds and two strands are separated. Two daughter strands are synthesized using two parental strands. The strands rewinds again in to two new double helixes. Each of these two helixes contain one parental strand and a new daughter strand. So, this is called semi-conservative replication. The replication process is complex and several enzymes are involved. The enzymes that forms phosphodiester bonds between adjacent nucleotide chain are called DNA polymerases.

DNA polymerase need a single strand template DNA to direct the synthesis of new strand. A short nucleic acid primer H bonded to the template DNA strand is required. Then DNA polymerase catalysis the formation of phosphodiester bond between the free 3’-hydroxyl group of the primer and the phosphate group of the nucleotide that is going to be incorporate.

DNA polymerase III involves in bulk respiration. It attached the deoxyribo nucleotide to the 3’-OH group of RNA/DNA primer. And DNA polymerase I it is responsible for removing RNA primers and replacing them with DNA. DNA synthesis always occurs from 5’ to 3’ direction.

In E.coli there are five proteins with polymerase activity. DNA pol I and III contributes for normal replication and DNA pol II, IV and V helps to repair the DNA and replication of damaged DNA.   

DNA Replication in Prokaryotes - Mechanism

In bacteria, most RNA molecules replicate as circular structure. So, due to the anti-parallel construction of DNA, Prokaryotic replication in DNA is bi-directional. So, the two strands should be separated to serve as templates. DNA helicase initiates the replication at origin of chromosomal replication (ori C) site by separating two strands of the circular double stranded DNA to make the replication bubble. Three types of DNA sequences in oriC functionally significant.

  • AT-rich region – HU (bacterial histone-like protein) and IHF (Integration host factor) proteins binds to this region. This causes the region wrap around the DnaA proteins and separate the AT rich region.
  • DnaA boxes – DnaA proteins binds to this region to initiate the replication and it stimulates the cooperative binding of an additional DnaA proteins to form a large complex.
  • GATC methylation sites
Prokaryotic DNA Replication
The photo credit goes to - Boumphreyfr / CC BY-SA (https://creativecommons.org/licenses/by-sa/3.0)

Separation of two strands make a V shape single strand region called replication folk. Many DNA helicase form a ring around one strand of DNA and propel the strand through the ring. So, the strands apart as the proteins move. Helicases leave a lot of ATP for energy. The DnaB of E.coli (Helicase of E.coli) propels one strand from 5’-3’ opening the strands of DNA ahead of the replication fork. DnaB ring cannot load on to SS-DNA on its own. It requires loading proteins called DnaC. DnaB separate the two DNA strands by breaking the H bonds between them. This generates positive supercoiling a head of each replication folk. In this case, Topoisomerase I, II and DNA gyrase travels ahead of the helicase and alleviates these supercoils.  There are SS binding proteins (SSB / Helix destabilizing proteins) binds to SS DNA and prevent reforming the Double Strand (DS) helical prematurely. DnaC protein released after assist the process. DNA Primase synthesize small stretches of RNA (10-12 nucleotides) that are complementary to the template strand. These are later removed and replaced with DNA. 

Replication Bubble

Due to the anti-parallel construction of DNA and the directionality of DNA polymerase III replication (5’-3’). There is a fundamental diference in replicating of two strands of DNA molecule. One strand is synthesized from 5’-3’ in the same direction that the replication folk is moving. This strand is called leading strand. The strand is occurring to the other direction from the moving of replication folk. The synthesis of this strand is discontinuous. The strand is called lagging strand. The short pieces of DNA synthesized during the lagging strand replication are called Okazaki Fragments. Synthesize of each of these fragments require a new RNA primer. 

DNA polymerase I removes the Short RNA primers and fills resulting gap with DNA. This has 5’- 3’ exonuclease activity which can be used to remove RNA primers and its 5’- 3’ polymerase activity to replace it with DNA. It can fill the gap using upstream okazaki fragments as the primer. Okazaki fragments should then be joint to form a continuous strand. DNA ligase forms a phosphodiester bonds between two separately synthesized DNA strands.

Opposite of the oriC is a pair of termination sequences called ter sequences (T1 and T2). The protein called tus protein (termination utilization substance) binds to these sequences. Then stop the movement of the replication forks. Finally, DNA ligase covalently links all four DNA strands.  So, DNA replication often results in two intertwined molecules. These intertwined molecules are known as catenanes. These are separated by the action of topoisomerases IV.

Termination

References

T.A.BROWN. (2010). GENE CLONING & DNA ANALYSIS (6th ed.). A John Wiley & Sons, Ltd,Publication.

Schleif, R. (2015). Genetics and Molecular Biology (2nd ed.). The Johns Hopkins University Press Baltimore and London.

Article By,

Pasindu Chamikara – Microbiologist

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