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).

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’.

• 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.

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.