DNA- The "Stuff" of Life Its replication and its unending repair

Watson and Crick published a second paper which suggested a hypothesis for the replication of DNA. That each strand of the DNA molecule could act as a template for the synthesis of opposite strand of the DNA molecule The question arises-Is DNA replication conservative, (The old strand is kept in tact, giving rise to two new strands), semi-conservative (gives rise to 2 strands an old strand and a new strand) or dispersive (new strands having both new and old strands)?

Matt Meselson and Frank Stahl (1958) demonstrated that replication was semi- conservative using radioactive nucleotides with dividing bacteria.

The bacteria was given radioactive nucleotides so that all the DNA was radioactive. Then once it was all radioactive, the bacteria was given normal radioactive and then allowed to replicate once. The DNA was then run on a agarose gel to see what banding pattern appeared.

conservative theory. The experiment was repeated but this time the bacteria was able to replicate twice. If replication was dispersive, only one band would be expected because all the DNA would be the same weight. What appeared though was two bands. One was radioactive and the other was not. This eliminated the dispersive theory. DNA replication is semiconservative theory. If replication was conservative, it is expected that two bands would appear, one radioactive and one normal. Only one radioactive band appeared instead of a two bands. This eliminated the

The following is a simple model for DNA replication.

-Because DNA is such a long molecule, replication must occur at the same time in many places. This is a micrograph, three replication bubbles are visible along the DNA cultured Chinese hamster cells. The arrows indicate the directions of DNA replication at the two ends of the bubble. There a number of proteins involved in DNA replication.

In prokaryotic cells, the chromosome is circular and not linear like eukaryotic cells. There is also only one origin for replication which attached to the plasma membrane. Replication of the chromosome occurs in both directions like eukaryotes. Prokaryotes have far fewer DNA base pairs than eukaryotes. E. coli has about 4.6 million base pairs whereas a human eukaryotic cell has 3 billion base pairs.

Notes about enzymes -DNA polymerase can only add to the 3' end of a nucleotide. This means that synthesis can only occur from the 5'->3' direction. -DNA polymerase must always have a nucleotide in front of it to hang the DNA nucleotide on. -Therefore an RNA primer must be laid down first and then replaced by DNA polymerase. RNA primase does this. -Helicases break the hydrogen bonds and gyrases relieve the stress. -Ligases suture DNA fragments together in the replicated molecule.

-Nucleotides are always added on as triphosphates. When the nucleotides are added then two phosphates are cleaved off making a pyrophosphate.

1. When it is time for the DNA to replicate, proteins attach to the places on the DNA molecule called origins of replication. On a prokaryotic cell there is one such site, but on a eukaryotic cell there are hundreds to thousands. 2. Helicases unwind the DNA strand and gyrases will nick one of the strands to relieve stress.

3. Single-stranded binding proteins stabilize the unwound parental DNA strand. 4. Primase will lay down a RNA primer so that the DNA polymerase can start base pairing because DNA polymerase must have something to bind to The molecule is replicated in both directions. One side is laid down

continuously, the other side is laid down in discontinuous fragments because and can only grow from the 5' to the 3' direction. These fragments are called Okazaki fragments. 5. The Okazaki fragments are joined together by ligase.

6. The RNA nucleotides are eventually replaced by DNA nucleotides when the DNA polymerase runs into them.

Once the DNA has been replicated, there is one problem. The usual replication machinery provides no way to complete the 5 ends, so repeated rounds of replication produce shorter DNA molecules. The solution to the problem is to put on the ends of the DNA, repetitive sequences of DNA. These sequences are noncoding sequences. These are called telomeres.

Telomeres only provide junk sequences so they prevent the erosion of genes are cells replicate. Note- in gametes, the shortening of telomeres would cause serious problems in multicellular organisms. If chromosomes of germ cells became shorter in every cell cycle, essential genes would eventually be missing from the gametes they produce. An enzyme called telomerase catalyzes the lengthening of telomeres in germ cells

Proofreading and Repairing DNA DNA polymerases proofread newly made DNA, replacing any incorrect nucleotides In mismatch repair of DNA, repair enzymes correct errors in base pairing DNA can be damaged by chemicals, radioactive emissions, X-rays, UV light, and certain molecules (in cigarette smoke for example) In nucleotide excision repair, a nuclease cuts out and replaces damaged stretches of DNA

The End