Molecular Genetics XIII

 

 

 

The Nature of DNA revisited

 

The structure of DNA suggests a copying mechanism as well as a means of information storage and retrieval. Watson and Crick’s second paper to Nature in 1953 describes how DNA might replicate itself and how the sequence of bases could code genetic information.

 

DNA workshop http://www.pbs.org/wgbh/aso/tryit/dna/index.html

 

 

DNA replication

 

During the S phase of interphase in cells destined to divide, the DNA is replicated. Watson and Crick suggested a semi-conservative model which was later accepted when put to experimental tests. The semi-conservative model means that when the double helix makes copies of itself, the two strands separate. The new daughter molecules will have one old (conserved) strand and one newly synthesized strand. 

 

Simple to follow, difficult to understand all its complexity. About 20 different enzymes are involved with replication. DNA replication is rapid and accurate.

 

Certain sequences of nucleotides (bases) signal an origin of replication. Bacterial, mitochondrial, and chloroplast DNA molecules will have one. Eukaryotic chromosomes may have hundreds to thousands of “ORIs”, depending on their size. The characteristic Y shape at the ends of the “replication bubble” is called the replication fork.

 

Steps and Rules in DNA synthesis

 

 1. The two strands of the DNA double helix must separate. An enzyme called helicase opens the DNA at the ORI. Single strand binding proteins keep the separated strands from rejoining.

 

2. An enzyme called DNA polymerase (one of three types) will catalyze the synthesis of the new strand according to base pair rules. DNA can add nucleotides (NTPs) only at a free 3’ OH group. In other words, DNA elongation is ALWAYS in a 5’ – 3’ direction.  DNA polymerase must have a free 3’ OH group to prime its synthesis and it cannot do this itself. So another enzyme, primase, places 10-20 RNA nucleotides onto the template strand. Primer formation must occur before any DNA nucleotides can be brought in.

 

3. On the leading strand, the DNAse synthesizes the new strand continuously as the replication fork opens further.

 

4. On the lagging strand, the DNAse must add nucleotides at the 3’ end AWAY from the opening replication fork. Synthesis on the lagging strand is therefore discontinuous. New primer must be laid down every 1000-2000 bases as the replication fork opens further. These islands of DNA nucleotides between primer segments are called Okazaki fragments.

 

5. When the DNA is completely replicated, the RNA primer segments must be replaced with DNA nucleotides. This is done with a different type of DNA polymerase.

 

6. Finally, an enzyme called ligase links the gaps in the 3’, 5’ sugar-phosphate backbone between the fragments.

 

Proofreading and repair

 

The rate of DNA replication is about 500 NTPs per second in prokaryotes. Eukaryotes are about 10 times slower (STILL, that’s 50 NTPs per second). The entire nuclear genome can be replicated in a few hours – that’s 6 billion NTPs!

 

Occasionally, the wrong base will be added to the growing chain at a rate of about one per ten thousand. That’s far too high for any organism. There is some proofreading capability in the DNAse itself and there are repair enzymes (about 50 of these) that can excise incorrect NTPs and replace them with the correct NTP.