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2 DNA replication

Learning Objectives

Upon completing this chapter, you will be able to:

  • Describe the general characteristics of bacterial DNA replication, including its speed and accuracy.
  • Identify the key enzymes and proteins involved in bacterial DNA replication and explain their specific roles (e.g., DNA polymerase III, helicase, primase, ligase, gyrase).
  • Outline the main steps in the initiation of DNA replication in bacteria, including the role of the origin of replication.
  • Explain the process of elongation, distinguishing between leading and lagging strand synthesis and the formation of Okazaki fragments.
  • Describe the termination process of DNA replication in bacteria with circular chromosomes.

Introduction to Bacterial DNA Replication

DNA replication is a fundamental process ensuring that genetic information is accurately copied before cell division. In bacteria, this process has been extensively studied, largely due to their relatively small genomes and the availability of mutants for genetic analysis. For instance, Escherichia coli possesses a genome of 4.6 million base pairs (Mbp) contained within a single circular chromosome. Remarkably, this entire chromosome is replicated in approximately 42 minutes. Replication initiates at a single origin of replication and proceeds bidirectionally (in both directions) around the circular chromosome. This rapid pace means that bacteria like E. coli add about 1000 nucleotides per second during replication, and this occurs with very few errors, highlighting the efficiency and precision of the molecular machinery involved.

The Machinery of Replication: Enzymes and Energy

DNA replication requires a coordinated effort of numerous proteins and enzymes. A central player is the enzyme DNA polymerase (DNA pol). Bacteria have several types of DNA polymerases:

  • DNA pol III: This is the primary enzyme responsible for synthesizing new DNA strands. It adds deoxyribonucleotides, one by one, that are complementary to the template strand. These nucleotides are added to the 3’-OH group of the growing DNA chain.
  • DNA pol I and DNA pol II: These polymerases are primarily involved in DNA repair processes, though DNA pol I also plays a role in removing RNA primers during replication.

The addition of nucleotides is an energy-requiring process. This energy is supplied by the deoxyribonucleotides themselves. Each incoming nucleotide is a deoxyribonucleoside triphosphate (dNTP), meaning it has three phosphate groups attached (similar to ATP, which stores energy for cellular processes). When a dNTP is added to the growing DNA strand, two of its phosphate groups (forming a pyrophosphate) are cleaved off. The energy released from breaking these high-energy phosphate bonds is used to form the covalent phosphodiester bond between the incoming nucleotide and the free 3’-OH group on the growing DNA strand (Figure 2.1).

Diagram of dGTP. In the center is deoxyribose which is a pentagon shaped sugar. The top point has an oxygen. Then, moving around the shape are carbons 1, 2, 3, and 4; carbon 5 is attached to carbon 4 but not in the ring. Attached to carbon 1 is a structure made of 2 carbon and nitrogen rings bound along their ends; this is guanine. Carbon 2 has only Hs attached to it. Carbon 3 has an H and an OH. Carbon 4 has an N and Carbon 5. Carbon 5 is attached to 3 phosphate groups in a row (labeled triphosphate). Each phosphate group is made of phosphorus attached to 4 oxygen atoms.
Figure x.x:  This structure shows the guanosine triphosphate deoxyribonucleotide that is incorporated into a growing DNA strand by cleaving the two end phosphate groups from the molecule and transferring the energy to the sugar phosphate bond. The other three nucleotides form analogous structures.

Initiation: Getting Replication Started

The initiation of replication is a precisely controlled process that begins at a specific nucleotide sequence on the chromosome called the origin of replication.

  • OriC: In E. coli, and most prokaryotes, there’s a single origin of replication named oriC. This region is approximately 245 base pairs long and is notably rich in adenine-thymine (AT) sequences. AT base pairs are held together by two hydrogen bonds, compared to the three hydrogen bonds in guanine-cytosine (GC) pairs, making AT-rich regions easier to separate.
  • Unwinding the DNA: Before replication can start, the DNA at oriC must be made accessible.
  • Bacterial chromosomal DNA is typically supercoiled and associated with histone-like proteins. Enzymes called topoisomerases are responsible for altering the shape and supercoiling of the chromosome. Topoisomerase II (also known as DNA gyrase) relaxes the supercoiled DNA.
  • Next, an enzyme called helicase separates the two DNA strands by breaking the hydrogen bonds between the complementary base pairs. This process requires energy from ATP hydrolysis.
  • Replication Forks and Bubble: As the DNA unwinds, Y-shaped structures called replication forks are formed. Since replication is bidirectional, two replication forks are established at oriC, moving in opposite directions. This creates a replication bubble, a region of unwound DNA visible under an electron microscope.
  • Stabilizing Single Strands: To prevent the separated single strands of DNA from reannealing (rewinding into a double helix), they are coated with single-stranded binding proteins (SSBPs).
  • The Need for a Primer: DNA polymerase III can only add nucleotides to an existing 3’-OH group; it cannot start a new DNA strand from scratch. This problem is solved by an RNA primer, a short sequence (typically 5-10 nucleotides) of RNA that is complementary to the template DNA. This primer provides the necessary free 3’-OH end.
  • Primase Action: The RNA primer is synthesized by an enzyme called RNA primase (an RNA polymerase). Unlike DNA polymerases, RNA polymerases can initiate RNA synthesis without requiring a pre-existing 3′-OH group.
  • DNA Pol III Begins Synthesis: Once the RNA primer is in place, DNA polymerase III can bind and begin adding DNA nucleotides, extending the primer and synthesizing a new DNA strand complementary to the template.

Elongation: Building the New DNA Strands

During elongation, new DNA strands are synthesized. DNA polymerase III adds nucleotides at its maximum rate (around 1000 nucleotides/second in E. coli). The anti-parallel nature of the DNA double helix (one strand runs 5’ to 3’, the other 3’ to 5’) presents a challenge because DNA pol III can only synthesize DNA in the 5’ to 3’ direction. This leads to different modes of synthesis for the two new strands:

  • Leading Strand: One template strand, oriented 3’ to 5’ relative to the movement of the replication fork, allows for continuous synthesis of the new DNA strand. DNA pol III can add nucleotides in the 5’ to 3’ direction moving towards the replication fork. This continuously synthesized strand is called the leading strand. It requires only one initial primer.
  • Lagging Strand: The other template strand is oriented 5’ to 3’ relative to the replication fork. To synthesize a new strand complementary to this template in the 5′ to 3′ direction, DNA pol III must move away from the replication fork. This results in discontinuous synthesis.
  • Okazaki Fragments: The lagging strand is synthesized in short, separate DNA segments called Okazaki fragments (named after their discoverers, Reiji and Tsuneko Okazaki). Each Okazaki fragment requires its own RNA primer, laid down by primase as more of the template strand is exposed by the advancing replication fork. DNA pol III then extends each primer.
  • The overall direction of synthesis for the lagging strand is 3’ to 5’ when considering the entire strand, but each individual Okazaki fragment is synthesized 5’ to 3’.
Diagram of DNA replication. A small inset at the top shows a double strand of DNA separated in the center forming a bubble; the DNA is double stranded on either side of the bubble. The origin of replication is in the midway point of the bubble. On the top strand a solid arrow points to the left from the origin; this is the leading strand. On the right of the origin of replication are short arrows pointing to the left; this is the lagging strand. On the bottom strand a solid arrow pointing to the right from the origin is labeled leading strand and short arrows pointing to the right on the other side of the origin are labeled lagging strands. A larger image shows just the left half of the bubble. The double stranded DNA is no the far left and is labeled 5’ for the top strand and 3’ for the bottom strand. An enzyme to the very far left I is labeled topoisomerase/gyrase. At the point where the double stranded regions splits is a triangle shape labeled helicase. Next to that are smaller shapes labeled single-stranded binding proteins. The top strand shows continuous synthesis of the leading strand; this is shown as a solid arrow under the top strand. The arrow has a 5’ at the right end and a 3’ at the left end. The template strand at the top has a 3’ at the right and a 5’ at the left. At the end of the arrow (near where the DNA is newly being separated by the helicase) is DNA polymerase 3 and a sliding clamp that span both strands. The bottom strand of DNA has more components. Just after the single stranded binding proteins is RNA primase which attaches RNA primer (shown as a green arrow). Further down the lagging strand template is an existing RNA primer with DNA polymerase III and a sliding clamp spanning primer and the template strand. The polymerase is building a new strand of DNA from the left side (5’) to the right side (3’). Further to the right is a long piece made of RNA primer, then new DNA, then RNA primer, then new DNA all connected. Each of the DNA/RNA combinations are okazaki fragments made in the discontinuous synthesis of the lagging strand. DNA polymerase I is attached to the RNA primer in the center and is replacing it with DNA nucleotides. DNA ligase then binds the individual strands of new DNA together. This is shown in a close-up as two double helices that have all the correct letters in place, but one is missing a connection between two of the nucleotides (this is called a single-stranded gap). DNA ligase forms this last bond and the gap is sealed.
Figure x.x:  At the origin of replication, topoisomerase II relaxes the supercoiled chromosome. Two replication forks are formed by the opening of the double-stranded DNA at the origin, and helicase separates the DNA strands, which are coated by single-stranded binding proteins to keep the strands separated. DNA replication occurs in both directions. An RNA primer complementary to the parental strand is synthesized by RNA primase and is elongated by DNA polymerase III through the addition of nucleotides to the 3’-OH end. On the leading strand, DNA is synthesized continuously, whereas on the lagging strand, DNA is synthesized in short stretches called Okazaki fragments. RNA primers within the lagging strand are removed by the exonuclease activity of DNA polymerase I, and the Okazaki fragments are joined by DNA ligase.

 

  • Sliding Clamp: A ring-shaped protein called the sliding clamp plays a vital role by holding DNA polymerase III firmly onto the DNA template as it synthesizes the new strand, increasing the processivity of the enzyme.
  • Managing Supercoiling: As helicase unwinds the DNA at the replication fork, positive supercoils can build up ahead of it. Topoisomerase II (DNA gyrase) alleviates this stress by introducing temporary nicks or double-strand breaks in the DNA, allowing it to untwist, and then resealing the DNA.
  • Primer Removal and Gap Filling: Once the Okazaki fragments are synthesized, the RNA primers must be removed and replaced with DNA.
  • The exonuclease activity of DNA polymerase I removes the RNA primers.
  • DNA polymerase I then fills in the resulting gaps with the correct DNA nucleotides.
  • Joining Fragments: After the gaps are filled, there are still nicks (breaks in the sugar-phosphate backbone) between the newly synthesized DNA (that replaced the primer) and the previously synthesized Okazaki fragment. The enzyme DNA ligase seals these nicks by catalyzing the formation of a phosphodiester bond between the 3’-OH end of one DNA fragment and the 5’ phosphate end of the adjacent fragment. This creates a continuous DNA strand.

Termination: Finishing Replication

Once the entire chromosome has been replicated, termination of DNA replication must occur. While initiation is well understood, the details of termination are less characterized.

  • Concatemers: In bacteria with circular chromosomes, the completion of bidirectional replication often results in two interlocked circular DNA molecules, known as concatenated chromosomes or concatemers. These must be separated so that each daughter cell receives a complete chromosome.
  • Topoisomerase IV: This separation is accomplished by the enzyme bacterial topoisomerase IV. It introduces double-stranded breaks into one of the DNA molecules, allowing the other to pass through, and then reseals the break.
  • Antimicrobial Targets: Because bacterial enzymes like DNA gyrase (topoisomerase II) and topoisomerase IV are distinct from their eukaryotic counterparts, they are excellent targets for antimicrobial drugs. The quinolone class of antibiotics, for example, specifically inhibits these bacterial topoisomerases, thereby blocking DNA replication and bacterial growth.

The Molecular Machinery Involved in Bacterial DNA Replication

The table below summarizes the key enzymes and proteins involved in bacterial DNA replication and their functions.

 

Enzyme or Factor Function
DNA pol I Exonuclease activity removes RNA primer and replaces it with newly synthesized DNA.
DNA pol III Main enzyme that adds nucleotides in the 5’ to 3’ direction.
Helicase Opens the DNA helix by breaking hydrogen bonds between the nitrogenous bases.
DNA Ligase Seals the gaps (nicks) between the Okazaki fragments on the lagging strand to create one continuous DNA strand.
Primase Synthesizes RNA primers needed to start replication.
Single-stranded binding proteins (SSBPs) Bind to single-stranded DNA to prevent hydrogen bonding between DNA strands, thus preventing the reforming of double-stranded DNA.
Sliding clamp Helps hold DNA pol III in place on the DNA template as nucleotides are being added.
Topoisomerase II (DNA gyrase) Relaxes supercoiled chromosome to make DNA more accessible for the initiation of replication; helps relieve stress on DNA when unwinding by causing breaks and then resealing the DNA.
Topoisomerase IV Introduces double-stranded breaks into concatenated chromosomes to release them from each other, and then reseals the DNA.

Chapter Summary

Bacterial DNA replication is a rapid, efficient, and highly accurate process essential for cell division. It begins at a specific origin of replication (oriC) where enzymes like helicase unwind the DNA, and DNA gyrase (topoisomerase II) relieves supercoiling. Single-stranded binding proteins stabilize the unwound DNA. Replication proceeds bidirectionally from two replication forks. DNA polymerase III, the main replicative enzyme, synthesizes new DNA in the 5’ to 3’ direction, requiring an RNA primer synthesized by primase. Synthesis is continuous on the leading strand and discontinuous (as Okazaki fragments) on the lagging strand. DNA polymerase I removes RNA primers and fills gaps, and DNA ligase seals nicks to join fragments. Finally, topoisomerase IV resolves concatenated circular chromosomes during termination. The unique enzymes involved in bacterial replication serve as targets for antimicrobial drugs.

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Microbial Genetics (Dr.B) Copyright © 2025 by Graham Boorse is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License, except where otherwise noted.