Chromosomes, Chromatin, DNA Replication and Repair

Edited Notes by Leigh Eisenman and Kevan Higgins

Experiments With Genetic Material

Griffith (1928)

Identified transforming principle. He worked with rough and smooth types of pneumonia bacteria. Smooth colonies have polysaccharide coating that makes them appear smooth

Griffith observed that something was transferred from the components of the killed smooth cell to the rough cell to change the rough cell's behavior. He called this unknown material that was transferred a transforming principle.

Avery, MacLeod, McCarthy (1944)

They were able to identify DNA as Griffiths transforming principle through the following experiment.

This showed that DNA, not protein, has ability to transform cells (for posterity's sake, they were actually mice, not rats)

Hershey and Chase (1950)

Proved DNA was hereditary material of cell. They worked with a bacterial virus (phage) comprised of protein coat and a DNA core. They wanted to find out which part of the phage (DNA or protein), produced new phages.

Watson and Crick (1953)

Watson and Crick discovered the "double helix" nature of DNA, a discovery which, along with Hershey and Chase, marked the beginning of molecular biology.

DNA Structure

DNA is obviously comprised of atoms, specifically N,C,H,O,and P. The cell needs not only these elements, but also other elements like Calcium, Magnesium, Manganese, Iron, Potassium, Sodium, Chloride. It also needs elements like Zinc, Cobalt, and Titanium in trace amounts.

DNA is built from nucleotides (adenine, thymine, cytosine, guanine). The basic structure consists of a sugar (deoxyribose) bonded to a base (A, T, G, C) at one position and bonded to a phosphate at a different position.

The bases protrude from the sugar-phosphate backbone and form hydrogen bonds with the bases on the complementary strand. These hydrogen bonds are important because they are not very strong so that the strands are easy to separate for duplication, yet they are strong enough to maintain the structure of the DNA.

Base Pairing: Adenine and thymine always bond with each other (A-T), while guanine and cytosine always bond to each other (G-C). Because of this relationship, one strand completely specifies the complementary strand and one strand is used to specify the other in replication. This complementary relationship provides a redundancy in the DNA which reduces the chance for error.

Directionality exists in DNA because the DNA strand has one 5' end and one 3' end. In a double stranded DNA the two strands run in opposite directions (they are antiparallel). The 3' end of one DNA strand lies at the same end of the double helix as the 5' end of the complementary strand. The directionality is the result of the DNA starting with a phosphate that is attached to the 5' position of the deoxyribose sugar of the first nucleotide. The second nucleotide in the chain is attached to the 3' position of that same sugar. The last nucleotide in the DNA has a free 3' end - nothing is attached to that (3') end.

The base pairs form a ladder and stack one on top of another along the inside of the helix, which serves to stabilize the structure.

The DNA structure is a right-handed double helix.Because the base pairs do not go directly through the middle of the helix, the sugar phosphate backbone on the outside of the helix are not symmetrical around the center of the helix. This results in either a smaller space between adjacent turns of the sugar-phosphate backbone around the outside of the helix, known as a minor groove, or a slightly larger space, known sensibly as a major groove.

When a cell is in interphase, the nucleus is said to be filled with chromatin, which represent the uncondensed metaphase chromosomes. In chromatin (and chromosomes) the ratio of DNA:histones:non-histones is 1:1:1, which means that DNA only really makes up about 1/3 of the material in chromosomes. The non-histone proteins are all of the chromatin associated proteins except the histones. These include RNA polymerases, transcription factors, and other enzymes that need to access the DNA.

The average human chromosome has about 2.5 feet of DNA. If all the nucleotides of the human genome were actual letters (A,C,G,T) and these were printed, they would fill 4,000 books of 500 pages each. That is roughly 3 billion nucleotides. All of that stuff fits into a cell roughly 1 millionth of an inch wide. This is accomplished because the DNA is highly compacted in the cells. The basic unit of packaging is known as a nucleosome. It consists of 8 histones molecules that form a core plus DNA, which is wrapped around this nucleosome core. The nucleosomes are packaged into higher order structures packing all the DNA very tightly together.

Histones themselves, since they have a very specific purpose, have been very conserved during evolution. There is almost no difference between a histone in a pea plant and a histone in a human.

The process of separating the two DNA strands is known as denaturing. When DNA are heated, the strands begin to come apart because the hydrogen bonds between base pairs break down. Because A-T base pairs have only two hydrogen bonds, they break apart first, followed by G-C base pairs which have three hydrogen bonds. This separation is very important for reading the DNA and also for duplicating it.

DNA Replication

Watson and Crick discovered the double helix structure and raised the question of how DNA was replicated. Three possibilities for DNA replication are:

Meselson and Stahl

Meselson and Stahl centrifuged the DNA to separate the molecules based on their densities.They followed the DNA for two generations as shown in the figure here.

Meselson & Stahl: DNA Replication is Semi-Conservative

Their results supported semiconservative replication as the correct model.

Replication occurs as the result of the enzyme DNA polymerase. As the DNA strands pull apart, the DNA polymerase moves along the template strand adding new base pairs to the strand being synthesized. When the strands denature in a given segment of DNA, a replication bubble is formed, having two replication forks at the ends where the strands continue coming apart. The point where replication begins is also known as the origin of replication. However, in eukaryotic cells, there is so much DNA that many different origins of replication are needed to be able to replicate all the DNA in the necessary time frame. When a DNA from one origin catches up to the DNA from another origin, they are joined an enzyme called a ligase.

When DNA is replicated the two strands come apart and tend to get intertwined, which can complicate many other biological processes including trying to divide the DNA equally between daughter cells. BUT! Thankfully there is an enzyme, topoisomerase, which changes the topology of DNA circles, in that it will break a DNA chain, take out the chain which is caught, and then rejoin the first chain.

All this unwinding of old strands and creation of new strands occurs at a remarkably fast rate. For each origin of replication, roughly 500-1000 nucleotides are added per second. That means that the strands are spinning at an incredibly fast rate: roughly 3000-6000 RPM. In the absence of topoisomerases, this process would burn up the cell.

Replication also contains built-in error checking. The frequency of errors is about 1 per 100 million bonds (1 x 10-8). Over the entire human genome, that works out to roughly 30 errors every single time the genome replicates. BUT! There are really only around three errors per replication because of DNA repair. If a repair enzyme finds a mistake, it can fix it, and it can tell which strand is wrong because it can tell which strand is the newly synthesized strand by at the extent of cytosine methylation. As DNAs exist in cells, many of the cytosines have a methyl group added to them by enzymes called methylases. A new DNA will have relatively few methylated cytosines because it has not been around long enough to have picked up that many methyl groups

Without DNA repair there can be some major problems. Xeroderma pigmentosum is a serious ailment caused by mutations in the gene for DNA repair. People with xp develop many skin tumors and other problems because of the number of errors in their DNA.

Biology 4: Genes and Society (1997, 1998)