CHROMOSOME STRUCTURE & DNA REPLICATION

Alu Sequences Can Be Mutagenic

Alu sequences are repetitive sequences dispersed all over the genome and can be within the genes or between the genes. Alu sequence is about 300 base pairs, it is repeated randomly 300000 times all over the genome. They do not have physiological functions as the rest of the DNA, But sometimes Alu sequences can be mutagenic factors (they can cause genetic diseases if they cause a rearrangement to the chromosome), how? Cholesterol level is controlled by the endocytosis process of blood cholesterol -which is carried on the LDL- by LDL receptors. When there is something wrong with the LDL receptor, then cholesterol will stay in the blood and its concentration will increase. The condition in which hypercholesterolemia is achieved is called familial hypercholesterolemia and this is because of defects in the LDL receptor which is responsible to take excess cholesterol by endocytosis and take it into the liver that metabolizes it. If there are defects in LDL receptor then LDL cholesterol will stay in blood causing many cardiovascular diseases. The figure represents the blood level of cholesterol under different physiological conditions. The normal concentration under normal physiological condition ranges from 100 to 250 or less mg/dl. A physiological condition called heterozygosity is in relation to familial hypercholesterolemia which is in relation to inheritance of the defect in LDL receptor gene. For each gene in somatic cells there are 2 copies; one copy is from the

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father and the other copy is from the mother. Regarding LDL receptor gene if there is one gene copy from one of the parents defected and the other is normal then that inheritance causes heterozygosity, so the concentration of cholesterol in the blood ranges from 300 to 500 mg/dl. If the two copies are defected from the parents and inherited to the offspring then there is homozygosity, so the LDL receptor is not functional at all and the concentration of cholesterol in the blood ranges from 600 to 1000 or more mg/dl. How do Alu sequences cause the defect in the LDL receptor gene?

The figure above represents LDL receptor gene, and the arrows represent Alu sequences within this gene. Most of them are in the introns and two of them are in the exons. Because Alu sequences in LDL receptor gene are in close proximity to each other and they are homologous, there

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will be unequal crossing over during meiosis which means there is a misalignment between the two chromosomes during the crossing over (exon 4 does not align with exon 4, and the same for Alu sequence and exon 5). The unequal crossing over results in the two products shown in the previous figure. Deletion of exon 5 in one of the chromosomal products and duplication of it in the other cause a defect in the LDL receptor and cause familial hypercholesterolemia.

Chromosome Structure
This is an electron microscopic picture of chromosome; we see beats on a string repeated in an organized form (equal distances between these beats). What is the structure of the beats? They are called nucleosomes which are found in eukaryotic genomes. It is very important to have nucleosomal structure for packaging of DNA on the chromosomes. This is a magnification of a nucleosome and it is composed of DNA and proteins (histones). The core of the nucleosome consists of negatively supercoiled DNA of 146 base pairs long wrapped around 8 histone protein molecules (there are 2 molecules of each type of these histone proteins

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[H2A, H2B, H3, H4] forming an octamer). The nucleosome as a whole is composed of DNA of 200 base pairs long wrapped around the histone octamer, and monomer histone molecule in the spacers that connects nucleosomes with each other. These DNA molecules wrapped around histones are resistant to nucleases, if you subject the chromosome to nuclease digestion and you run an electrophoresis for the DNA produced after nuclease digestion, what will you see in the picture of electrophoresis? We will see fragments of DNA and the length of a fragment is 200 base pairs because nuclease will cut between nucleosomes and will be unable to digest the DNA which is wrapped around the histones, so we will see a ladder-like picture of fragments. Histones What is the significance of histones? Since the whole backbone of DNA is negative and histones are positively charged so they stabilize the genomic structure by these electrostatic interactions. The sequences of the different types of histones are homologous and they are homologous also in every species all over the evolutionary tree. Why are there different types of histones? They might have different amounts of positive charges that do more stabilization for the DNA on the chromosome. Also there will be different responses of these histones to modification.
General information about histones:

1) Histones are small proteins; each histone gene has one exon and no introns. 2) They are positively charged because they are rich in arginine and lysine (positively charged amino acids because they are basic, and they are basic because they have a basic group with very high pka

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[12 for Arg R group and about 10.5 for Lys R group], they will not be titrated at physiological pH so they stay positively charged). 3) They also interact with negatively charged DNA. 4) They can be extensively modified. Modification of Histones Modification means addition or removal of groups. It is one of the important mechanisms to regulate gene expression. When a gene being expressed, the DNA must unfold, the wrapped DNA around histones must be unfolded and must be exposed so no histones will stay in a gene or in a DNA when it is expressed. When it is not expressed all of them are packaged with each other. By receiving the signal for expression everything will be unfolded and the chromosome or the DNA will be exposed without any nucleosome structures on it for other proteins to come and activate its expression. So how will DNA be unfolded from the nucleosomal structures? By modification to delete the positive charges of histones, then there will be uncoiling or unfolding of DNA to be ready for expression.
There are different processes of modification of histones:

1) Phosphorylation of histones gives negative charge. In which amino acid residue of histones? Serine or Tyrosine that have the hydroxyl group in R groups. 2) Poly (ADP) Ribosylation. 3) Methylation: methyl groups on some reactive groups to mask them and that affects their function. 4) Acetylation: Adding an acetyl group to affect active group in the histone. Hypoacetylation: There is an increase in the positive charge of histones that leads to increase the hold of histones to DNA so DNA is not exposed to proteins that cause expression. Hypoacetylation is associated with repression of gene expression and transcription. Hypoacetylation is caused by the

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enzyme histone deacetylase which is activated by retinoblastoma protein (RB). Hyperacetylation: to mask positive charges on histones so there will be destabilization of the nucleosomal structures and the DNA will be unfolded and hyperacetylated. Hyperacetylation is associated with activation of transcription and gene expression. Hyperacetylation is activated by some transcriptional factors. Telomeres

The ends of chromosomes are called telomeres. They are DNA sequences rich in G and C bases. A telomere is a satellite repeated sequence and ranges from 1 Kb to more than 12 Kb. They are important since they are considered as caps to protect the ends of the chromosome from shortening and from karyotypic rearrangements during DNA replication.

The young cell has a very long telomere while the aged cell has a short telomere. During replication always there is a loss of DNA sequences from the ends of the chromosome and this is why in aged cells there are no more telomeres. Genes will be also degraded from the ends of the chromosomes and that will cause aging and death of those cells. So one of

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the reasons of aging is degradation of telomeres causing no more telomeres to protect our chromosomes from degradation during DNA replication. What causes telomeres always in young cells to have full-length of sequences? An enzyme called telomerase. So every round of DNA replication, telomerase works on telomeres and extends them to the proper length, with age telomerase activity decreases. What is the reason that cancer cells are not aged and they are always active and do not die? Because they have very active telomerase enzyme that always works on telomere and increases the length of it.

Packaging of DNA into chromosomes

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The previous figures represent packaging of DNA into chromosomes. In order to accommodate the very large DNA molecule in the very small nucleus there must be a mechanism of packaging of DNA into chromosome. The Doctor played a video about DNA http://www.youtube.com/watch?v=gbSIBhFwQ4s). packaging (link:

DNA passes through many steps of packaging, it starts as naked DNA then it forms the nucleosomal structure, then these nucleosomes fold on each other forming structures that refold on each other. There is an increase in the thickness (diameter) through the steps of packaging (there is an increase in the diameter of the helix related to the diameter of the structure with nucleosomes). After refolding of nucleosomes with each other, the folded nucleosomes bind to a protein called scaffold protein. Many scaffold structures after that step refold on each other to finally form the chromatid or the chromosome structure.

DNA Replication

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This is the mammalian cell cycle, in S phase DNA synthesis or DNA replication take place and histones are synthesized (after G1 phase). In G1 phase there is a rapid growth and preparation for DNA synthesis (DNA synthesis requires a lot of proteins and enzymes) so in this phase there is synthesis of the required proteins and enzymes for DNA replication. Before cell division DNA must be replicated. DNA replication is semiconservative. What does it mean? DNA is doublestranded, it is important to be a double-stranded structure for DNA replication because each strand will act as a template for the synthesis of the new molecules of double-stranded DNA that resemble the parental DNA molecule, if DNA was not double-stranded then that goal would not be achieved. As you see in the figure when the double-stranded DNA replicates, the two strands must unfold and each strand acts as a template, and that will continue till you reach the end of the DNA molecule. So each daughter (new) DNA molecule has the original parental DNA strand as a template and a newly synthesized strand which is complementary and antiparallel to the parental DNA template. And because of this the two daughter DNA molecules exactly resemble the parental DNA molecule, so the DNA replication is semiconservative. Our DNA is very big and requires hours to finish its replication. And to replicate in few hours there must be many sites where the chromosome

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starts the replication from (there is no one single site to initiate the replication on each chromosome). Every site at which the replication starts from is called the origin of replication. Prokaryotes have only one origin of replication because they have small genome. Our genome has multiple origins of replication in order to replicate the entire DNA in few hours (if there was one origin of replication, it would take years to finish DNA replication).

On each origin of replication there is melting of the double-stranded DNA into single-stranded and the replication starts bidirectionally to form replication folds, then these replication folds diffuse with each other on the chromosome and finish the replication in the required time.

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To start DNA replication the nucleosomes must be removed by hyperacetylation through signals to make DNA exposed to proteins. Among these proteins is a protein called dnaA protein which binds to the origin of replication and causes melting of DNA. When does DNA melt? After dnaA proteins molecules aggregate with each other. Then dnaB and dnaC proteins bind into the melted DNA region in the single-stranded DNA molecule. dnaB protein helps the DNA to unwind in order for the coming proteins to reach DNA templates and continue replication.

Done by: Amjad Habeb

LECTURE OF 3 OCTOBER Page 11 of 11

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