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Bacteria do not have the end-replication problem, because its DNA is circular.   In eukaryotes, the chromosome ends are called telomeres which have at least two functions:

• to protect chromosomes from fusing with each other.
• to solve the end-replication problem.  

The procedure to solve the end-replication problem is outlined in Figure 7-C-1.  Mechanism of the telomere extension by telomerase is explained in Figure 7-C-2.

 Figure 1.  Telomerase and telomere extension.  To extend the length of a telomere, the telomerase first extends its longer strand.  Then, using the same mechanism as synthesizing the lagging strand, the shorter strand is extended.

Figure 2.  The mechanism of telomere extension by telomerase.

 

Synthesis of the lagging strand requires a short primer which will be removed.  At the extreme end of a chromosome, there is no way to synthesize this region when the last primer is removed.  Therefore, the lagging strand is always shorter than its template by at least the length of the primer.  This is the so-called "end-replication problem".
Bacteria do not have the end-replication problem, because its DNA is circular.   In eukaryotes, the chromosome ends are called telomeres which have at least two functions:

• to protect chromosomes from fusing with each other.
• to solve the end-replication problem.  

The procedure to solve the end-replication problem is outlined in Figure 7-C-1.  Mechanism of the telomere extension by telomerase is explained in Figure 7-C-2.

 

Figure 7-C-1.  Telomerase and telomere extension.  To extend the length of a telomere, the telomerase first extends its longer strand.  Then, using the same mechanism as synthesizing the lagging strand, the shorter strand is extended.

Figure 7-C-2.  The mechanism of telomere extension by telomerase.


In a human chromosome, the telomere is about 10 to 15 kb in length, composed of the tandem repeat sequence: TTAGGG.  The telomerase (web link) contains an essential RNA component which is complementary to the telomere repeat sequence.  Hence, the internal RNA can serve as the template for synthesizing DNA.  Through telomerase translocation, a telomere may be extended by many repeats.

Aging
In the absence of telomerase, the telomere will become shorter after each cell division.  When it reaches a certain length, the cell may cease to divide and die.  Therefore, telomerase plays a critical role in the aging process.

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There is a major difference between DNA polymerase and RNA polymerase: the RNA polymerase can synthesize a new strand whereas the DNA polymerase can only extend an existing strand.  Therefore, to synthesize a DNA molecule, a short RNA molecule (~ 5 - 12 nucleotides) must be synthesize first by a special enzyme.  The initiating RNA molecule is known as a primer, and the enzyme is called primase.

In addition to DNA polymerase and primase, DNA replication requires helicase and single strand binding protein (SSB protein).  The role of helicase is to unwind the duplex DNA.  SSB proteins can bind to both separated strands, preventing them from annealing (reconstitution of double-stranded DNA from single strands).
The replication mechanisms in both bacteria and eukaryotes are similar.  However, eukaryotic DNA polymerases do not contain a subunit similar to the E. coli b subunit.  They use a separate protein called proliferating cell nuclear antigen (PCNA) to clamp the DNA.

DNA polymerases can extend nucleic acid strands only in the 5' to 3' direction.  However, in the direction of a growing fork, only one strand is from 5' to 3'.  This strand (the leading strand) can be synthesized continuously.  The other strand (the lagging strand), whose 5' to 3' direction is opposite to the movement of a growing fork, should be synthesized discontinuously.


Figure 3.  Steps in the synthesis of the lagging strand.
(a) Comparison between the leading strand and the lagging strand.
(b) The primase first synthesizes a new primer which is about 10 nucleotides in length.  The distance between two primers is about 1000-2000 nucleotides in bacteria, and about 100-200 nucleotides in eukaryotic cells.
(c)  DNA polymerase elongates the new primer in the 5' to 3' direction until it reaches the 5' end of a neighboring primer.  The newly synthesized DNA is called an Okazaki fragment.
(d) In E. coli, DNA polymerase I has the 5' to 3' exonuclease activity, which is used to remove a primer.
(e) DNA ligase joins adjacent Okazaki fragments.
The whole lagging strand is synthesized by repeating steps (b) to (e).

source: http://www.web-books.com/MoBio/Free/Ch7B3.htm

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Figure 1.  The structure formed by two b subunits of  the E. coli DNA polymerase III .  This structure can clamp a DNA molecule and slide with the core polymerase along the DNA molecule.

DNA polymerases

E. Coli

Three types of DNA polymerases exist in E. coli: I, II and III.  The DNA polymerase I is used to fill the gap between DNA fragments of the lagging strand.  It is also the major enzyme for gap filling during DNA repair.  The DNA polymerase II is encoded by the PolB gene, which is involved in the SOS response to DNA damage.   DNA replication is mainly carried out by the DNA polymerase III. 

The DNA polymerase III consists of several subunits, with a total molecular weight exceeding 600kD.  Among them, a, e, and q subunits constitute the core polymerase.  The major role of other subunits is to keep the enzyme from falling off the template strand.  As shown in Figure 7-B-1, two b subunits can form a donut-shaped structure to clamp a DNA molecule in its center, and slide with the core polymerase along the DNA molecule. This allows continuous polymerization of up to 5 x 10⁵ nucleotides.  In the absence of b subunits, the core polymerase would fall off the template strand after synthesizing 10-50 nucleotides. 

Mammals

There are five types of DNA polymerases in mammalian cells: a, b, g, d, and e.  The g subunit is located in the mitochondria, responsible for the replication of mtDNA.  Other subunits are located in the nucleus.  Their major roles are given below:

• a: synthesis of lagging strand.
• b: DNA repair.
• d: synthesis of leading strand.
• e: DNA repair.

 source: http://www.web-books.com/MoBio/Free/Ch7B.htm

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DNA replication is triggered by the expression of all required proteins, such as DNA polymerase, DNA primase, and cyclin.  In yeast, the transcription factor regulating the expression of these proteins is called MCB binding factor.  In mammals, the corresponding transcription factor is E2F.

Table 1.  Genes regulated by the yeast MCB binding factor.

 
Figure 1.  Schematic drawing of the DNA replication process.  O1, O2, and O3 are replication origins, each serving a region called replicon (R1, R2, and R3).  DNA replication involves unwinding of the double helix.  New strands are synthesized by DNA polymerases using the old strands as template.  Unwinding of a DNA molecule looks like a "fork" growing in one direction.  The region being replicated looks like a bubble called the "replication bubble" (in red).
In E. coli, movement of the growing fork is about 1000 bp per second.  It takes about 42 minutes to duplicate the entire genomic DNA.  In eukaryotic DNA, the fork movement is only about 100 bp per second.  This is probably due to the association of DNA with histones, which may hinder the fork movement.  In humans, replication of the entire genome requires about 8 hours.  In fruit flies, it takes only 3 - 4 minutes.
 
Figure 2.  Sequences of bacterial and yeast replication origins.  (a) The bacterial replication origin contains three homologous 13-mers and four homologous 9-mers.  (b) The yeast replication origin is called autonomously replicating sequence (ARS).  The "A" region is absolutely necessary, while B1, B2, and B3 can increase the replication efficiency. 
Based on computer analysis, the consensus sequence of human replication origin is  WAWTTDDWWWDHWGWHMAWTT, where W = A or T; D = A, G or T ; H = A, C or T, and M = A or C.
Note that "A" and "T" dominate the replication origin.  This is because the A-T pair is linked by two hydrogen bonds while C-T pair by three hydrogen bonds.  Therefore, the region dominated by "A" and "T" should be easier to unwind.

source:  http://www.web-books.com/MoBio/Free/Ch7A.htm


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