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Wednesday, October 31, 2007

DNA Polymerase I and the Synthesis of Okazaki Fragments

Slightly modified from Horton et al. (2006) ...

During DNA replication, a molecular machine called a replisome forms at the replication fork where the two strands of DNA are separating. The replisome contains activities that separate the strands and hold them apart for synthesis by the replisome version of DNA polymerase, called DNA polymerase III in bacteria. The complex has two sliding clamps that bind the complex to the strands of DNA so that DNA replication is highly processive.

DNA polymerases catalyze chain elongation exclusively in the 5′ → 3′ direction. Since the two strands of DNA are antiparallel, synthesis using one template strand occurs in the same direction as fork movement, but synthesis using the other template strand occurs in the direction opposite fork movement. The new strand formed by polymerization in the same direction as fork movement is called the leading strand. The new strand formed by polymerization in the opposite direction is called the lagging strand.


Deoxyribonucleic Acid (DNA)
Recall that the replisome contains a DNA polymerase III holoenzyme dimer with two core complexes that can catalyze polymerization. One of these is responsible for synthesis of the leading strand, and the other is responsible for synthesis of the lagging strand.

Here's a video showing the entire process (source unknown, please contact me if you know who made this video). The details of one of the important steps are presented below the fold.

A. Lagging-Strand Synthesis Is Discontinuous

The leading strand is synthesized as one continuous polynucleotide, beginning at the origin and ending at the termination site. In contrast, the lagging strand is synthesized discontinuously in short pieces in the direction opposite fork movement. These pieces of lagging strand are then joined by a separate reaction.

The short pieces of lagging-strand DNA are named Okazaki fragments in honor of their discoverer, Reiji Okazaki. The overall mechanism of DNA replication is called semidiscontinuous to emphasize the different mechanisms for replicating each strand.

B. Each Okazaki Fragment Begins with an RNA Primer

It is clear that lagging-strand synthesis is discontinuous, but it is not obvious how synthesis of each Okazaki fragment is initiated. The problem is that no DNA polymerase can begin polymerization de novo; it can only add nucleotides to existing polymers. This limitation presents no difficulty for leading-strand synthesis since once DNA synthesis is under way nucleotides are continuously added to a growing chain. However, on the lagging strand, the synthesis of each Okazaki fragment requires a new initiation event. This is accomplished by making short pieces of RNA at the replication fork. These RNA primers are complementary to the lagging strand template. Each primer is extended from its 3′ end by DNA polymerase I to form an Okazaki fragment, as shown in the Figure. (Synthesis of the leading strand also begins with an RNA primer, but only one primer is required to initiate synthesis of the entire strand.)

The use of short RNA primers gets around the limitation imposed by the mechanism of DNA polymerase, namely, that it cannot initiate DNA synthesis de novo. The primers are synthesized by a DNA dependent RNA polymerase enzyme called primase—the product of the dnaG gene in E. coli. The three-dimensional crystal structure of the DnaG catalytic domain revealed its folding and active site are distinct from the well studied polymerases, suggesting that it may employ a novel enzyme mechanism. Primase is part of a larger complex called the primosome that contains many other polypeptides in addition to primase. The primosome, along with DNA polymerase III, is part of the replisome.

As the replication fork progresses, the parental DNA is unwound, and more and more single-stranded DNA becomes exposed. About once every second, primase catalyzes the synthesis of a short RNA primer using this single-stranded DNA as a template. The primers are only a few nucleotides in length. Since the replication fork advances at a rate of about 1000 nucleotides per second, one primer is synthesized for approximately every 1000 nucleotides that are incorporated. DNA polymerase III catalyzes synthesis of DNA in the 5′ → 3′ direction by extending each short RNA primer.

C. Okazaki Fragments Are Joined by the Action of DNA Polymerase I and DNA Ligase

Okazaki fragments are eventually joined to produce a continuous strand of DNA. The reaction proceeds in three steps: removal of the RNA primer, synthesis of replacement DNA, and sealing of the adjacent DNA fragments. The steps are carried out by the combined action of DNA polymerase I and DNA ligase.

DNA polymerase I of E. coli was discovered by Arthur Kornberg about 45 years ago. It was the first enzyme to be found that could catalyze DNA synthesis using a template strand. In a single polypeptide, DNA polymerase I contains the activities found in the DNA polymerase III holoenzyme: 5′ → 3′ polymerase activity and 3′ → 5′ proofreading exonuclease activity. In addition, DNA polymerase I has 5′ → 3′ exonuclease activity, an activity not found in DNA polymerase III.

DNA polymerase I can be cleaved with certain proteolytic enzymes to generate a small fragment that contains the 5′ → 3′ exonuclease activity and a larger fragment that retains the polymerization and proofreading activities. The larger fragment consists of the C-terminal 605 amino acid residues, and the smaller fragment contains the remaining N-terminal 323 residues. The large fragment, known as the Klenow fragment, is widely used for DNA sequencing and many other techniques that require DNA synthesis without 5′ → 3′ degradation. In addition, many studies of the mechanisms of DNA synthesis and proofreading use the Klenow fragment as a model for more complicated DNA polymerases.

The Figure (right) shows the structure of the Klenow fragment complexed with a fragment of DNA containing a mismatched terminal base pair. The 3′ end of the nascent strand is positioned at the 3′ → 5′ exonuclease site of the enzyme. During polymerization, the template strand occupies the groove at the top of the structure and at least 10 bp of double-stranded DNA are bound by the enzyme, as shown in the figure. Many of the amino acid residues involved in binding DNA are similar in all DNA polymerases, although the enzymes may be otherwise quite different in three-dimensional structure and amino acid sequence.

The unique 5′ → 3′ exonuclease activity of DNA polymerase I removes the RNA primer at the beginning of each Okazaki fragment. (Since it is not part of the Klenow fragment, the 5′ → 3′ exonuclease is not shown in the Figure above, but it would be located at the top of the structure, next to the groove that accommodates the template strand.) As the primer is removed, the polymerase synthesizes DNA to fill in the region between Okazaki fragments, a process called nick translation (see Figure below). In nick translation, DNA polymerase I recognizes and binds to the DNA chain. In this way, the enzyme moves the nick along the lagging strand. After completing 10 or 12 cycles of hydrolysis and polymerization, DNA polymerase I dissociates from the DNA, leaving behind two Okazaki fragments that are separated by a nick in the phosphodiester backbone. The removal of RNA primers by
DNA polymerase I is an essential part of DNA replication because the final product must consist entirely of double-stranded DNA.

The last step in the synthesis of the lagging strand of DNA is the formation of a phosphodiester linkage between the 3′-hydroxyl group at the end of one Okazaki fragment and the 5′-phosphate group of an adjacent Okazaki fragment. This step is catalyzed by DNA ligase. The DNA ligases in eukaryotic cells and in bacteriophage-infected cells require ATP as a cosubstrate. In contrast, E. coli DNA ligase uses NAD+ as a cosubstrate. NAD+ is the source of the nucleotidyl group that is transferred, first to the enzyme and then to the DNA, to create an ADP-DNA intermediate.

[©Laurence A. Moran and Pearson/Prentice Hall]

Horton, H.R., Moran, L.A., Scrimgeour, K.G., Perry, M.D. and Rawn, J.D. (2006) Principles of Biochemistry. Pearson/Prentice Hall, Upper Saddle River, NJ (USA)


Nick Woolridge said...

The animation is by Drew Berry, of the Walter and Eliza Hill Research Institute in Melbourne:


Nick Woolridge said...

...And I should mention that the rather "purple' music is not original; Drew works with a sound designer to add much more usefully evocative sound effects in the originals.

Larry, if you want, I am in the MSB (on the main floor) and have a DVD containing many of Drew's animations at full quality that you can borrow...


Larry Moran said...

Thanks, Nicolas. I wonder if Drew knew John Wilkins.

Anonymous said...

Very recent work, particularly by Ken Marians' group, provides evidence that the leading strand has the ability to also be synthesized in a discontinuous manner, for example to reinitiate downstream when faced with a polymerase-incompatible lesion in the leading strand template. There is also good evidence (unlike the depiction in the animated cartoon) than when the leading strand template is blocked, lagging strand synthesis becomes decoupled and can proceed past the furthest extent of leading strand synthesis for several hundreds of nt. This is important for template-switch repair processes.

There are also some fun questioners (crackpots?) out there still wondering as to whether in vivo leading strand synthesis really is continuous. See for example:

Wang TC. Discontinuous or semi-discontinuous DNA replication in Escherichia coli? Bioessays. 2005 Jun;27(6):633-6.

It's tempting to suggest that biochemistry professors should "Teach the Controversy"(tm) about this important question of continuous or discontinuous leading strand synthesis. :)


L. Ba said...

thanks a lot. I am learning for german abitur and I had a question in my mind, whether it is the polymerase which is removing the primer for okazaki fragments or the exonuclease. I didn't know that (in this case) the polymerase I is the exonuclease, because I could't find any information in my books about exonuclease, even not in biology from campbell.
But you wrote about polymerase I having this exonuclease activity. If I am right, eucaryonts do not have polymerase I but polymerase III. So how do they remove the primers?

Anonymous said...

The primer is erased by a special DNA repair enzyme (an RNAse H) that recognizes an RNA strand in an RNA/DNA helix and fragments it.

Unknown said...

Klenow (3′→ 5′ exo-) is a mesophilic DNA polymerase deficient in both proofreading (3′→ 5′) and nick-translation (5′→ 3′) nuclease activities, and that displays a moderate strand displacement activity during DNA synthesis. The protein is expressed as a truncated product of the E.coli PolA gene and contains the D355A and E357A mutations.

Unknown said...

I understand that DNA polymerase can't start synthesis de novo.I am unclear of how DNA pol 1 removes the RNA primer and replaces it with DNA on the leading strand if the primer is now removed. Could you please shed some light on the subject