DNA: Replication

The hereditary material in a cell is coded in the sequence of the heterocyclic amines of DNA. There are normally 46 strands of DNA called chromosomes in human cells. Specific regions, called genes, on each chromosome contain the hereditary information which distinguishes individuals from each other. The genes also contain the coded information required for the synthesis of proteins and enzymes needed for the normal functions of the cells. Bacterial cells may have 1000 genes, while the human cell contains more than a million genes. A single E. coli (bacteria) chromosome of double helical DNA consists of 3.4 million base pairs.

Introduction

Prior to cell division, the DNA material in the original cell must be duplicated so that after cell division, each new cell contains the full amount of DNA material. The process of DNA duplication is usually called replication. The replication is termed semiconservative since each new cell contains one strand of original DNA and one newly synthesized strand of DNA. The original polynucleotide strand of DNA serves as a template to guide the synthesis of the new complementary polynucleotide of DNA. A template is a guide that may be used for example, by a carpenter to cut intricate designs in wood.

The DNA single strand template serves to guide the synthesis of a complementary strand of DNA. DNA polymerase III is an example of this process. More explanation in the next panel.

DNA Replication Process

Several enzymes and proteins are involved with the replication of DNA. At a specific point, the double helix of DNA is caused to unwind possibly in response to an initial synthesis of a short RNA strand using the enzyme helicase. Proteins are available to hold the unwound DNA strands in position. Each strand of DNA then serves as a template to guide the synthesis of its complementary strand of DNA. DNA polymerase III is used to join the appropriate nucleotide units together. The replication process is shown in graphic on the left.

Template #1 guides the formation of a new complementary #2 strand. The DNA template guides the formation of a DNA complementary strand - not an exact copy of itself. For example looking at template # 2, this process occurs because the heterocyclic amine, adenine (A), codes or guides the incorporation of only thymine (T) to synthesize new DNA #1. The replication of DNA is guided by the base pairing principle so that no other heterocyclic amine nucleotide can hydrogen bond and fit correctly with cytosine. The next heterocyclic amine, cytosine (C), guides the incorporation of guanine (G) while similar arguments apply to the other bases. Exactly the opposite reaction occurs using template #2 (far right margin) where cytosine (C) guides the incorporation of guanine (G) to form a new complementary #2 strand.

It is so important that the cells duplicate the DNA genetic material exactly, that the sequence of newly synthesized nucleotides is checked by two different polymerase enzymes. The second enzyme can check for and actually correct any mistake of mismatched base pairs in the sequence. The mismatched nucleotides are hydrolyzed and cut out and new correct ones are inserted.

DNA Replication

Although details of DNA replication is not thoroughly understood, because so many molecules are involved in the process. This example focuses on the bacteriophage T7 DNA replication complex because it consists of relatively few proteins. The mechanism of T7 DNA replication is a good model for other DNA replication. This molecule is based on the recent work of Doublie, et al. (1998). In the graphic below, the DNA polymerase enzyme is shown with a short section of DNA. The green color represents the DNA template, while the magenta color represents the newly synthesized DNA.

In the close up, guanine triphosphate nucleotide is shown on the active site, guided by the cytosine in the template matching through hydrogen bonds. Only a few of the enzyme protein side chain interactions with the nucleotide are shown. Magnesium ions are also active in stabilizing the triphosphate through ionic interactions. Eventually the two of the phosphates are hydrolyzed and the remaining phosphate is bonded in a phosphate ester bond to the deoxyribose on the end of the newly forming DNA chain.

References

1. Doublie S., Tabor S., Long A., Richardson C., and Ellenberger T. (1998). Crystal Structure of a Bacteriophage T7 DNA Replication complex at 2.2 A Resolution. Nature 391: 251-258.

Problems

1. In the green ribbon form of the enzyme, on the outside there are several series where the ribbons lie side by side. What protein structure is this?
2. On the inside of the "clamp", what protein structures are visible?
3. Using a single strand of DNA, write the sequence of heterocyclic amines to make its complementary strand.
   Template: A C T A G G

Answers

1. These are beta pleated sheets in the tertiary structure.
2. There are numerous alpha helices that line the "hole" in the tertiary structure.
3. Complementary T G A T C C

Outside Links

• Link to: Great Animation of entire DNA Repllication - John Kyrk
• More details: E. coli DNA Polymerase III Beta Subunit The Sliding DNA Clamp © David Marcey, 1997
• Reference: Kong, X-P., Onrust, R., O'Donnell, M., and J. Kuriyan (1992). Three-Dimensional Structure of the Beta Subunit of E. coli DNA Polymerase III Holoenzyme: A Sliding DNA Clamp. Cell 69: 425-437.
• More Details: The Bacteriophage T7 DNA Replication Complex, Michael E. Ward and David Marcey.

Contributors

• Charles Ophardt, Professor Emeritus, Elmhurst College; Virtual Chembook