Overview of DNA replication and its enzymes

Question 1

Briefly describe the main function of DNA polymerase

Answer 1

DNA is replicated by enzymes known as DNA polymerases. They use ssDNA as templates to catalyze the synthesis of the complementary strand. The nucleotides that are selected are complementary to the nucleotides on the template strand (according to Watson-Crick base pairs), so that the newly synthesized strand can form a double helix with the template. Nearly all DNA polymerases can only add a nucleotide that is donated from a nucleoside triphosphate to the 3’-OH group of the complementary base pair so that the DNA chain is extended in the 5’-3’ direction.

Question 2

Explain what a replication bubble is and how it was discovered

Answer 2

One of the early indications of how chromosomes replicate was obtained through the autoradiography of replicating DNA. Autoradiograms of circular chromosomes grown in a medium containing [H^3] thymidine show the presence of replication eyes or bubbles. These 0(theta) structures (it resembled theta) indicate that dsDNA replicates by the progressive separation of its two parental strands accompanied by the synthesis of their complementary strands to yield two semiconservatively replicated duplex daughter strands. DNA replication involving theta structures is known as theta replication.

Question 3

What is a replication fork

Answer 3

A branch point in a replication bubble at which DNA synthesis occurs is called a replication fork. A replication bubble may contain one or two replication forks (unidirectional or bidirectional replication).

Question 4

What is the role of DNA gyrase

Answer 4

The parent DNA strands unwind at the replication fork. Naturally occurring DNA’s negative supercoiling promotes DNA unwinding, but only to the extent of about 5% of its duplex turns. However, in prokaryotes negative supercoils may be introduced into DNA through the action of a type IIA topoisomerase called DNA gyrase, at the expense of ATP hydrolysis.

Question 5

Explain semidiscontinuous replication

Answer 5

The two parent strands are replicated in different ways. The newly synthesized strand that extends in its 5’-3’ in the direction of the replication fork movement, so the leading strand is continuously synthesized in the 5’-3’ direction. The other newly synthesized strand, called the lagging strand is also synthesized in the 5’-3’ direction but discontinuously as Okazaki fragments. The Okazaki fragments are covalently joined together after their synthesis by DNA ligase.

Question 6

List the enzymes that are involved in DNA replication and the order that they appear

Answer 6

DNA replication is a complex process that involves a variety of enzymes in the process. DNA topoisomerases, enzymes known as helicases separate the DNA strands at the replication fork, proteins that prevent them from reannealing before they are replicated, enzymes that synthesize RNA primers, a DNA polymerase, an enzyme to remove the RNA primers and an enzyme to covalently link successive Okazaki fragments.

Question 7

Explain unwinding of DNA strands

Answer 7

Helicases separate the DNA strands by unwinding them at the replication fork. Proteins work together to perform this task- DnaB protein (from dnaB gene), Rep helicase and single-strand binding proteins. This process requires energy- is driven by ATP hydrolysis. A helicase functions by translocating along one strand of DNA so as to separate the strands in its path. This requires free energy and is therefore driven by NTP hydrolysis.

Question 8

Give a brief desciption of helicases

Answer 8

Helicases are classified into 6 superfamilies that vary in characteristics such as their direction of translocation along the DNA strand (5’-3’ or 3’-5’) and whether they function as as hexameric rings or dimers. DnaB protein is a hexameric helicase that separates the strands of dsDNA by translocating along the lagging strand template in the 5’-3’ direction while hydrolyzing A/G/CTP.

Question 9

Describe the hexameric helicases

Answer 9

There are two families of hexameric helicases: the RecA family and AAA+ family. RecA translocates in the 5’-3’ direction and occurs mainly in eubacteria and their phages (DnaB is a part of the RecA family. AAA+ hexameric helicases translocates in the 3’-5’ direction and occur mainly in archaea, eukaryotes and their viruses.

Question 10

Describe rep helicase

Answer 10

Two other helicases: Rep helicase and PriA protein are involved in the replication of various E.coli phage DNAs and they also participate in certain aspects of E.coli DNA replication. Both protein translocate in the 3’-5’ direction while hydrolyzing ATP. Rep helicase consists of a monomer but dimerizes when bound to ssDNA or dsDNA. Both subunits of the Rep dimer bind to DNA such that when the DNA binds to one subunit it strongly inhibits DNA binding to the other. Therefore the “active, rolling mechanism” is used for Rep-mediated DNA unwinding in which the 2 subunits of the dimer alternate in binding dsDNA and the 3’ end of ssDNA at the ssDNA/dsDNA junction. The 2 subunits “walk” up the DNA while unwinding it (and using ATP) via a subunit switching mechanism in which one of the subunits are bound to the ssDNA and the other subunit (free) binds to dsDNA and unwinds it, it displaces its 5’-starting strand while remaining bound to its 3’-starting stand. The other subunit from the 3’-starting ssDNA is released and then this subunit binds to the new end of the dsDNA and unwinds it, thereby continuing the cycle.

Question 11

Describe the function of single-strand binding protein

Answer 11

Separated DNA strands can anneal rapidly- this is prevented by the binding of single-strand binding proteins. It also prevents ssDNA from forming intramolecular helical structures( helical stems) and protects it from nucleases. The single-strand binding proteins must be removed from ssDNA before replication.

Question 12

Describe RNA primers

Answer 12

The analysis of Okazaki fragments showed that their 5’ ends consist of RNA. E.coli consists has 2 enzymes that can catalyze the formation of RNA primers- RNA primase and RNA polymerase. Primase is insensitive to the RNA polymerase inhibitor rifampicin. Rifampicin inhibits only leading strand synthesis, this indicated that primase initiates the Okazaki fragment primers.

Question 13

Describe primase

Answer 13

Primase is an enzyme that catalyzes the synthesis of RNA primers. They are closely associated with helicases. E.coli primase (DnaG) forms a noncovalent complex with DnaB. DNA helicases translocate along the lagging strand in the 5’-3’,direction, therefore primase must reverse its direction of travel in order to synthesize a RNA primer in its 5’-3’ direction. DnaG is held to the RNA primed site by its association with SSB, can synthesize up to 60nt primers. Primases tend to initiate synthesize at specific 3-nt sequences on the template.

Question 14

Describe the main function of DNA polymerase I

Answer 14

DNA polymerase I couples dNTPs on the DNA template in a reaction that involves the nucleophilic attack of the growing DNA chain’s 3’-OH group on the alpha-phosphoryl of an incoming nucleoside triphosphate. PPi is released and is hydrolysed by pyrophosphatase. This provides this reaction with the energy that it requires. This is the polymerase activity of DNA polymerase I.

Question 15

What are the other functions of DNA polymerase I

Answer 15

It also has 2 hydrolytic activities:
> 3’-5’ exonuclease activity- if pol I incorporates the wrong nucleotide at the end of a growing chain, the polymerase activity is inhibited and the 3’-5’ exonucleases excise this nucleotide. The polymerase activity then resumes replication. Polymerase I therefore has the ability to proofread a DNA chain so it can correct any mistakes.
> 5’-3’ exonuclease activity- the pol I 5’-3’ exonuclease binds to dsDNA at single-strand nicks and cleaves the DNA in a base paired region beyond the nick such that the DNA is excised as mononucleotides or oligonucleotides. This can be used to remove an RNA primer.

Question 16

Explain the distribution of the functions of Pol I in its structure

Answer 16

The 5’-3’ exonuclease activity of Pol I is separate from its 3’-5’ exonuclease activity and its polymerase activity. Proteases can cleave DNA polymerase I in 2 fragments- a larger C-terminal or klenow fragment, which contains 3’-5’ exonuclease activity and the polymerase activity and a smaller N-terminal fragment that contains 5’-3’ exonuclease activity. Polymerase I contains 3 active sites on a single polypeptide chain.

Question 17

Describe the structure of the klenow fragment

Answer 17

The klenow fragment consists of 2 domains- the smaller domain contains the 3’-5’ exonuclease site and the larger domain contains the polymerase site. The fragments domains are known as “fingers”, “thumb” and “palm”, The “fingers” binds the incoming nucleotide, the “thumb” guides the newly formed dsDNA as it leaves the active site and the “palm” contains the active site.

Question 18

Explain DNA polymerases catalytic mechanism

Answer 18

All DNA polymerases share a common catalytic mechanism. Their active sites all contain 2 metal ions, usually Mg2+. Metal ion A activates the primer’s 3’OH group for a nucleophilic attack on the incoming dNTPs alpha-phosphate group and metal ion B orients and stabilizes its bound negatively charged triphosphate group.

Question 19

Explain nick translation

Answer 19

Pol Is combined polymerase and 5’-3’ exonuclease activities can replace nucleotides on the 5’-end of single-strand nicked DNA. It moves the nick towards the 3’-end without changing the molecule.

Question 20

Describe DNA polymerase III holoenzyme

Answer 20

The pol III core has the subunit composition of alphaEtheta, its alpha subunit contains polymerase activity and has a similar structure as pol I- the fingers, thumb and palm domains. The catalytic properties of Pol III core resemble those of Pol I, except it cannot be replicate primed ssDNA or nicked dsDNA. It is the main replicative polymerase in E.coli.

Question 21

Describe DNA polymerase IIIs beta-subunit

Answer 21

The beta-subunit forms a C-shaped homodimer that associates to form a donut-like structure- this is known as the sliding clamp or the beta clamp. The sliding clamps central opening has a diameter or 35A which is larger than the diameters of B- and ADNA. It forms a closed ring around DNA, which prevents it from escaping.

Question 22

Describe the function of DNA ligase

Answer 22

Pol I replaces the RNA primers with DNA through nick translation. The resulting single-strand nicks between adjacent Okazaki fragments as well as the nick on circular DNA after leading strand synthesis, are sealed in a reaction catalyzed by DNA ligase.

Question 23

Describe the mechanism of this function

Answer 23

The free energy that is required for this reaction is acquired from the hydrolysis of NAD+ to NMN+ + AMP or ATP to AMP+PPi. The E.coli enzyme LigA utilizes NAD+ and catalyzes this reaction. This is a 3 step reaction: the adenylyl group of NAD+ is transferred to the lys residue of the enzyme, then the adenylyl group is transferred from the enzyme to the 5’-phosphoryl end of the nicked DNA and then DNA ligase catalyzes the formation of a phosphodiester bond by attack of the 3’-OH group on the 5’-phosphoryl group thereby sealing the nick and releasing AMP.
ATP-requiring DNA ligases(eukaryotes and bacteriophage T4) release PPi in the 1st step of the reaction rather than NMN+. T4 ligase can at high concentrations link two duplex DNAs (blunt end ligation)