DNA Replication

The double-helical structure of DNA immediately suggests a simple mechanism for the accurate duplication of the genetic information stored in DNA

  
 

Each strand contains all of the information necessary to specify the sequence of its complementary strand.

When the two strands are pulled apart, by the action of various proteins, each single strand can be used as a template upon which to synthesize of a new strand.

The basic monomer of nucleic acid polymerization is a nucleotide triphosphate.  For DNA these are the deoxynucleotides, deoxyguanidine triphosphate (dGTP), deoxycytodine triphosphate (dCTP), deoxyadenosine triphosphate (dATP) and deoxythymidine triphosphate (dTTP).

 

Polymerization involves the reaction of an incoming deoxynucleotide triphosphate (dNTP) with the 3' hydroxy group of the pre-existing polynucleotide chain.

The reaction releases pyrophosphate (P-P) and forms a phosphodiester bond.

The 3' OH of the added dNTP becomes the new 3' OH of the polynucleotide.

  
 

The sequence of a polynucleotide is always written in the 5' to 3' direction, just as the sequence of a polypeptide is always written from N-terminus to C-terminus.

Both DNA and RNA polymerases synthesize nucleic acids by adding nucleotides to the 3' -OH end of a molecule.

 
  • How is the directionality (polarity) of polynucleotide chain determine?
  • Why are nucleotide triphophates used to synthesize DNA and RNA? 
  • Why are amino acid triphosphates not used in protein synthesis, do you think?
 

In vivo DNA replication:  DNA replication is a complicated process – the steps are mediated by various proteins.

In the bacterium Escherichia coli over 100 genes are involved in DNA replication and repair.

Replication begins at specific sequences along the DNA strand, known as origins of replication or origins for short.

Origin sequences are recognized by specific proteins. The binding of these proteins initiates the assembly of an origin recognition complex an ORC).

In the laboratory, DNA is generally heated to 94-100°C in order to insure complete denaturation.

In the cell, various proteins act on the DNA to locally denature (unwind) the DNA to form a replication bubble. 

  

 
A multiprotein complex will assemble at each end of the replication bubble, these structures are known replication forks.
 
  Using a single replication origin and two replication forks, a growing E. coli can replicate its ~4,700,000 base pairs of DNA in ~40 minutes.
 

Each replication fork moves along the DNA synthesizing ~1000 base pairs of DNA per second

Synthesis is an accurate process; the polymerase makes about one error for every 10,000 base pairs it synthesizes.

Most of the errors it makes are quickly recognized, since they lead to the formation of a base mismatch.  When a mismatched base pair is formed, the DNA polymerase recognizes it, reverses and removes it (using an exonuclease activity), and that resynthesizes it, correctly.

This process is known as proof-reading; the proof-reading activity of the DNA polymerase complex reduces the total DNA synthesis error rate to ~1 error per 1,000,000,000 (109) base pairs synthesized.

 
  • Assume that there is a mutation that alters the proof-reading function of the DNA polymerase complex - what will have to cell?
  • How does the DNA polymerase recognize a mismatched base pair?
  • Why do you need to denature the DNA double-helix to copy it?
  • How does the DNA polymerase complex know where to start replicating DNA?  
 

DNA replication and molecular machines:  The process of nucleic acid replication involves a number of molecule machines, we will consider only one as an example - the clamp loader. 

Once DNA replication begins it is important that the polymerase complex remains attached to the DNA.

This is accomplished by the assembly of a circular protein, known as a sliding clamp, around the DNA double helix.

 
 

The clamp is added to DNA by a protein known as a clamp loader.  Once closed around the DNA the clamp diffuses freely along the DNA molecule, but it cannot leave the DNA. 

It diffuses along, but not away from, the DNA molecule.

The rest of the polymerase complex is attached to the clamp.

 

This keeps polymerase complex from drifting away from the DNA.

The attached clamp-polymerase complex now assembles many nucleotides into a new DNA strand before it falls off -- this behavior is termed processive.

 
  • Explain why, in the absence of a clamp loader, the polymerase could diffuse away from the DNA?
  • Why might this be "bad"?
  • How do you think the clamp is removed?  
 
DNA-dependent, DNA polymerases cannot start a new DNA chain de novo or from nothing. They can only add nucleotides to a pre-existing DNA strand.
 

When the DNA helix opens there are no pre-existing DNA strand for the DNA polymerase to extend.

So, how does synthesis start?

 
 

The answer is that a short RNA molecule, known as a primer, complementary to one strand of the open DNA, is synthesized by a DNA-dependent, RNA polymerase - this enzyme is sometimes known as primase.

Initially two primers are synthesized, one complementary to each of the two DNA strands.
 

DNA polymerase synthesizes DNA onto the ends of these primers in the standard 5' to 3' manner. These first primers define the leading strands of DNA synthesis.

As the DNA forks moves, more DNA is unwound, and new primers have to be synthesized to insure replication of the lagging strands. No new primers have to be synthesized on the leading strand.

 
 

Later in the DNA replication process, the RNA primers are digested away, replaced with DNA, and joined together to produce a single, uninterrupted replicated strand. 

 
 

Eukaryotic cells can contain more than 1000 times the DNA found in a typical bacterial cell.  At the same time, and the eukaryotic replication complex synthesizes DNA at about 5% of the rate of the bacterial complex. 

While it may take a bacterial cell about 1.5 x 103 seconds to replicate its 3 x 106 base pair chromosome; it can take many hours for compensate by using many more replication origins.  

So even they may contain 1000 times more DNA than E. coli, DNA replication can still be completed in as little as 30 minutes.
 
  • Why is a RNA primer needed? 
  • Is an RNA primer needed to make an mRNA, do you think?
  • Why is only a single RNA primer need to synthesize the leading strands, but multiple primers are need to synthesize the lagging strands?  
  • During the replication of a single DNA molecule, how many leading and lagging strands are there?
Use Wikipedia | revised 10 March 2005