Functions and Dysfunctions of Genomic Regulation

Question 1

LOs #1-2 Functions and dysfunctions of genomic regulation

Answer 1

A. Recognize and describe the physical and functional organization of the Eukaryotic Genome which includes:

i. DNA building blocks
ii. DNA packaging and Histones
iii. Chromosome structure
iv. Genes

B. Describe the basic processes of gene regulation and expression

i. DNA replication – Identify and describe the basic steps of DNA replication and enzymes involved
ii. Transcription
i. Identify different types of RNA
ii. Describe gene regulatory elements
iii. Know the basic RNA synthesis process including basic RNA processing reactions

Question 2

Describe ‘The Central Dogma’

Answer 2

The central dogma of molecular biology explains the flow of genetic information, from DNA to RNA, to make a functional product, a protein

The central dogma states that the pattern of information that occurs most frequently in our cells is:

• From existing DNA to make new DNA (DNA replication)
• From DNA to make new RNA (transcription)
• From RNA to make new proteins (translation).

Question 3

Describe DNA structure

Answer 3

DNA contains the structural blueprint for all genetic instructions.

The genetic code contained within the DNA is composed of four “letters” or bases:

• Purines—adenine (A) and guanine (G)
• Pyrimidines—cytosine (C) and thymidine (T)

The DNA have double-helix structure.

• DNA backbone comprises five-carbon sugar (pentose) molecules bound to a nucleoside (A, G, C, or T). The pentose molecules are also asymmetrically joined to phosphate groups by phosphodiester bonds. “Phosphate-deoxyribose backbone”
• Hydrogen bonds between complementary (G:C or A:T) nucleotides (a nucleoside linked to a sugar and one or more phosphate groups) interact to stabilize and form the double-helix structure.

Question 4

Describe the double helix in detail.

Answer 4

DNA exists as a double helix,

10 nucleotide pairs per helical turn.

Major and minor grooves in each turn.

Each of the two helical strands is composed of the sugar phosphate backbone with attached bases and is connected to a complementary strand by hydrogen bonding.

The pairing of the nucleotide bases occurs such that adenine binds with thymine and guanine with cytosine.

Forms of the DNA

• B form - 10 nucleotide pairs per turn, right-handed
• A form -11 nucleotide pairs per turn, right-handed
• Z form - left-handed B form - actively transcribed DNA

Question 5

Describe DNA structural organization

Answer 5

DNA is a high molecular weight double-stranded polymer.

Deoxyribonucleotides joined by covalent phosphodiester bonds.

The phosphodiester bonds are bonds that form between the 3′-OH groups of the deoxyribose sugar on one nucleotide with the 5′ phosphate groups on the adjacent nucleotide.

The phosphodiester linkages between individual deoxynucleotides are directional in nature.

The 5′ phosphate group of one nucleotide is bound to the 3′ hydroxyl group of the next nucleotide.

The two complementary strands of DNA double helix run in antiparallel directions. The 5′ end of one strand is base-paired with the 3′ end of the other strand.

The 5′ end of one strand is base-paired with the 3′ end of the other strand. This primary structure is stabilized by noncovalent interactions - Hydrogen bonds

Nucleotide bases on one strand form these bonds with nucleotide bases on the opposite strand.

Adenine forms two hydrogen bonds with thymine, while guanine and cytosine are connected by three hydrogen bonds.

Base pairing in the interior of the helix stabilizes the interior of the double-stranded DNA because the stacked bases repel each other due to their hydrophobic nature.

The hydrogen bonds between bases can be made and broken easily, allowing DNA to undergo accurate replication and repair

Question 6

Describe DNA packing and Histones.

Answer 6

The DNA is packaged into a protein-DNA structure called chromatin.

Allow DNA to fit in the nucleus.

Chromatin consists of very long double-stranded DNA molecules, small basic proteins called histones, as well as smaller amounts of nonhistone proteins, and a small quantity of RNA.

Nucleosomes are the fundamental organization upon which the higher order packing of chromatin is built

Question 7

What are Nucleosomes?

Answer 7

The nucleosome core consists of a complex of eight histone proteins with double-stranded DNA wound around it.

Histones are a heterogeneous group of closely related arginine- and lysine-rich basic proteins.

These positively charged amino acids help histones to bind tightly to the negatively charged sugar phosphate backbone of DNA. Histones provide for the compaction of chromatin.

Each nucleosome core consists two molecules each of histone H2A, H2B, H3, and H4.

Linker histone H1 separates each nucleosome

Question 8

Describe chromosomal structure.

Answer 8

Chromosome structure varies with the cell cycle, from the loose thread-like appearance in the growth (G1) phase to the tightly compacted state observed during division (M) phase. Chromosomes have three elements as individual units:

• Telomeres are hexameric DNA repeats [(TTAGGG)n] found at the ends of chromosomes that serve to protect the chromosome from degradation
• Centromeres serve as “handles,” which allow mitotic spindles to attach to the chromosome during cell division. The centromere also serves as a boundary that separates the two arms.
• Multiple origins of replication, in order for DNA in chromosomes to replicate, a specific nucleotide sequence acts as a DNA replication origin They are dispersed throughout its length. At the origin of replication, there is an association of sequence-specific, double-stranded DNA-binding proteins with a series of direct repeat DNA sequences.

Question 9

What is a gene?

Answer 9

A gene is the complete sequence region necessary for generating a functional product/protein.

The gene area encompasses promoters and control regions necessary for the transcription, processing, and translation.

About 2% of the genome encodes instructions for the synthesis of proteins.

Genes are concentrated in random areas along the genome, with vast expanses of noncoding DNA between them.

The coding regions of a gene are called exons.

The noncoding regions are called introns

Question 10

Describe Characteristics of DNA replication/synthesis

Answer 10

1. Semiconservative with respect to parental strand

When DNA is replicated during the process of cell division, one parent or original strand of DNA is distributed to each daughter duplex in combination with a newly synthesized strand with an antiparallel orientation.

At the end of the process, each of the two daughter strands has half new DNA and half old DNA

2. Bidirectional with multiple origins of replication

DNA replication is bidirectional and starts in several different locations at once.

Replication begins at several sites on linear DNA and is completed by the end of DNA synthesis (S) phase of the cell cycle. As replication nears completion, “bubbles” of newly replicated DNA come together forming two new molecules

3. Primed by short stretches of RNA

DNA replication requires a short stretch of ribonucleic acid (RNA) for the initiation of the process.

DNA polymerases cannot initiate synthesis of a complementary strand of DNA on a totally single-stranded template.

DNA primase, synthesizes short stretches of RNA that are complementary and antiparallel to the DNA template.

The RNA primer is later removed.

Chain elongation is carried out by DNA polymerases by the addition of deoxyribonucleotides to the 3′ end of the growing chain.

The sequence of nucleotides that are added is dictated by the base sequence of the template (or coding) strand with which the incoming nucleotides are paired

4. Semidiscontinuous with respect to the synthesis of new DNA

All DNA polymerases function in the same manner: They “read” a parental strand 3′ to 5′ and synthesize a complementary antiparallel new strand 5′ to 3′.

DNA polymerase synthesizes one strand in the 5′ to 3′ continuously called the leading strand.

The opposite strand is synthesized 5′ to 3′, but discontinuously, this strand is called the discontinuous or lagging strand

The DNA synthesized on the lagging strand as short fragments (100 to 200 nucleotides) is called the Okazaki fragments.

Although overall chain growth occurs at the base of the replication fork, synthesis of the lagging strand occurs discontinuously in the opposite direction but with exclusive 5′ to 3′ polarity

Question 11

What are the proteins involved in DNA replication for each step?

Answer 11

1. Parental DNA Strand Separation

DNA helicases are a class of motor proteins required to unwind short segments of the parental duplex DNA.

Topoisomerases remove tthe supertwisting of DNA

Single-stranded DNA-binding proteins prevent premature annealing of the single-stranded DNA to double-stranded DNA

2. DNA Synthesis/Chain Elongation

DNA primases initiate the synthesis of an RNA molecule essential for priming DNA synthesis on both the leading and the lagging strands.

DNA polymerases function as a complex to initiate DNA synthesis and chain elongation by adding new nucleotides. the also have exonuclease activity, or proofreading ability, that allows them to remove nucleotides that are not part of the double helix.

3. DNA ligation

DNA ligase is an enzyme that catalyzes the sealing of nicks (breaks) remaining in the DNA after DNA polymerase fills the gaps left by RNA primers. DNA ligase is required to create the final phosphodiester bond between the adjacent nucleotides on a strand of DNA

Question 12

What are Telomeres?

Answer 12

The telomere, a protective repetitive stretch of DNA complexed with protein at the end of a chromosome, shortens with every cell division.

The lagging strands of replicated telomeres undergo shortening after the removal of the last RNA primer from the 5′ ends during each successive cycle of cell division.

The gap cannot be filled in due to the lack of a primer, which lead to shortening of the telomeres with every cell division.

Telomere shortening is recognized as and is a part of the normal aging process.

The telomere maintenance enzyme, telomerase, is an RNA-dependent DNA polymerase, which adds TTAGGG repeats to the ends of the chromosomes

Question 13

Describe transcription.

Answer 13

Types of RNA:

Ribosomal RNA - Structure and function of ribosomes

Transfer RNA - Carry amino acids to ribosomes for protein synthesis

Messenger RNA Carrier of genetic information from genes to ribosomes for protein synthesis

MicroRNA - regulate mRNA stability and downregulate gene expression

Question 14

LOs #3-4 Functions and Dysfunctions of Genomic Regulation

Answer 14

2. DNA Damage and Mutations

A. Describe and differentiate between the different types of DNA damage i. Spontaneous DNA damage ii. Physical agents induced damage (Radiation induced damage) iii. Chemical agents induced damage (Direct and indirect)

B. Recognize and describe the different types of genomic alterations i. Chromosomal mutations ii. Gene amplification iii. Transposons iv. Single-nucleotide polymorphisms

Question 15

Describe DNA Damage.

Answer 15

Spontaneous: – The cell environment is not static, metabolic activity, and DNA replication is not perfect – Basal mutation rate : 2×10-10 mutations per bp per replication – Happen on daily bases – DNA is surrounded with active chemistry – Most frequent two examples are depurination and deamination

• Physical agents: Radiation – Ionizing – None Ionizing
• Chemical agents: Direct and in direct

Radiation interaction with matter can last from10−18𝑠 to generations (years). • But the biology is created by a chemical event initiated by the deposition of the radiation energy

Radiation-Induced DNA Damage – Direct (bond break) – Indirect (H2O hydrolysis) and free radicals formation

• UV-Induced DNA Damage – Formation of the pyrimidine Dimers

Chemically induced DNA Damage – Two types • Agents that act directly to modify DNA • Agents that require metabolic activation

Question 16

Describe genomic alterations.

Answer 16

• Happen normally and through DNA damage and repair
• Can be good – evolution and normal function such as immune response
• Can be bad – disease and death
• Can be nothing

• Four major types: – Chromosomal mutations – Gene amplification – Transposons – Single-nucleotide polymorphisms

Question 17

What are the 4 types of chromosomal mutations?

Answer 17

Deletions

Translocation

Duplication

Inversion

Example: Translocations can cause Down Syndrome

Question 18

What are Transposons?

Answer 18

Transposons

• Jumping genes
• Mobile sequences of DNA that can change position within the genome of a single cell
• Copy/Cut > past
• Example :Hemophilia A is a hereditary bleeding disorder caused by a lack of blood clotting factor VIII. Without enough factor VIII, the blood cannot clot properly to control bleeding.

– Transposon L1 inserted into the factor VIII gene renders factor VIII ineffective

Question 19

What are Single-nucleotide polymorphisms?

Answer 19

Single-nucleotide polymorphism (SNP) is a common inherited change in a single base pair that occurs in at least 1-5% of the population

• Not a mutation (uncommon alteration in the DNA) but can act as one
• SNPs occur once every 1000 to 2000 nucleotides
• SNPs are used as markers in the mapping of genomes
• Polymorphism may be associated increase susceptibility to disease
• Can also be used in drug development

Question 20

Describe gene amplification

Answer 20

Question 21

LOs #5-7 Functions and Dysfunctions of Genomic Regulation

Answer 21

A. Understand the importance of DNA repair and its outcomes

B. Recognize and describe the different types of DNA repair mechanisms and understand the types of damages repaired. i. Direct repair ii. Base excision repair iii. Nucleotide excision repair (NER) iv. Mismatch excision repair (MER) v. Recombination repair a. nonhomologous end joining (NHEJ) b. homologous recombination vi. Transcription-coupled repair (TCR) vii. Trans-lesion

C. Discuss examples of DNA repair associated disorders and diseases i. Xeroderma pigmentosum ii. Hereditary nonpolyposis colorectal cancers iii. Cockayne syndrome iv. BRCA mutations and breast cancer

Question 22

Describe the DNA Direct Repair mechanism.

Answer 22

Direct repair

• The damage

– UV damage (adjacent pyrimidines)

– alkylating mutagens

Question 23

Describe the DNA Base excision repair (BER) mechanism.

Answer 23

The damage

– Single-base mismatches and small, nondistorting alterations

– Spontaneous depurination and spontaneous deamination

– Uracil, 8-oxoguanine, 3-methyladenine, and abasic sites

Question 24

Describe the DNA Nucleotide excision repair (NER) mechanism.

Answer 24

Nucleotide excision repair (NER)

• The damage – Chemical adducts – Alteration in DNA shape in the local area – UV, BPDEguanine adducts, and cisplatin adducts
• Xeroderma pigmentosum – genetic disorder of DNA repair in which patients carry mutations in the nucleotide repair enzymes mostly an endonuclease – High sensitivity to sunlight – pigmentary skin change – autosomal recessive – prone to developing melanomas and squamous cell carcinomas. – defects in the NER complex XP proteins (XP-A through XP-G)

Question 25

Describe the DNA Transcription-coupled repair (TCR)(form of NER) mechanism.

Answer 25

The Damage – RNA polymerase stalls at a lesion in the DNA – TCR proteins called ERCC-6 and ERCC-8 recognize the stalled RNA polymerase and recruit other repair proteins

• Cockayne syndrome – Autosomal recessive – Mutant genes ERCC6 and ERCC8. – Neurologic delay – Photosensitivity – Progeria (premature aging). – Commonly hearing loss and eye abnormalities

Question 26

Describe the DNA Mismatch excision repair (MER) mechanism.

Answer 26

The Damage – During DNA replication – Mismatched nucleotide occasionally gets inserted into the newly synthesized daughter strand

• Hereditary nonpolyposis colorectal cancers – Lynch syndrome – Mutations in one of the alleles of genes in the MER complex – Accumulation of unrepaired DNA damage lead to genomic instability -> Cancer

Question 27

Describe the DNA Recombination repair mechanisms

Answer 27

• The Damage – Double strand breaks – Radiation and chemical
• Two Type: – Nonhomologous end joining (NHEJ)- Chromosomal translocation - neoplastic chromosomal rearrangements
• Homologous recombination (HR) -Needs a template - After DNA replication and before cell division

Recombination repair: Homologous recombination (HR) in breast cancer: • Susceptibility genes (BRCA1 and BRCA2) associated with hereditary breast cancer encode proteins that facilitate homologous recombination • Five-fold increase risk of developing breast Cancer and other Cancers

Question 28

LOs #8-9 Functions and Dysfunctions of Genomic Regulation

Answer 28

Genetic Code and Mutations A. Understand the genetic code and its features B. Recognize the different types of point mutations C. Understand and discuss the four different categories of mutation i. silent mutation ii. missense mutation iii. nonsense mutation iv. frameshift mutation

Discuss Epigenetics and its role in genomic dysfunction and disease A. Examples of epigenetic modifications i. Gene methylation ii. Histone acetylation B. Cancer as an example

Question 29

Describe Genetic Code and Mutations

Answer 29

The genetic code is standard but not universal

• Exceptions in mitochondria
• Codons AUA and AUU code for Met (instead of Ile), and AGA and AGG act as stop codons (instead of Arg). The standard stop codon, UGA, codes for Trp instead in mitochondria

Mutation in the coding region also called open reading frame (ORF) lead to different amino acid being incorporated into the protein

• This leads to different outcomes categorized into four type of point mutations:

– Silent mutation • The codon containing the changed base may code for the same amino acid. • For example, if the serine codon UCA is given a different third base “U” to become UCU, it still codes for serine. This is termed a “silent” mutation.

– Missense mutation

– Nonsense mutation

– Frameshift mutation

Question 30

What is a missense mutation?

Answer 30

Missense mutation

– The codon containing the changed base may code for a different amino acid.

– For example, if the serine codon UCA is given a different first base “C” to become CCA, it will code for a different amino acid, in this case, proline.

– The substitution of an incorrect amino acid is called a “missense” mutation.

– Sickle cell anemia: • missense mutation in the β-globin gene in which GAG a glutamate is changed to GTG a valine, giving rise to HbS. • Alters the conformation of hemoglobin, the deoxy form • Mutated hemoglobin molecules aggregate, forming rigid rod-like structures, and cause the deformation of red blood cells into a sickle-like shape. It have poor oxygen-carrying capacity and tend to clog capillaries, thus restricting blood supply to various tissues. • High prevalence in African Americans • Symptoms include: Anemia, Episodes of pain, Frequent infections, Delayed growth or puberty

Question 31

What is a nonsense mutation?

Answer 31

Nonsense mutation

– The codon containing the changed base may become a termination codon.

– For example, if the serine codon UCA is given a different second base “A” to become UAA, the new codon causes termination of translation

– Production of truncated protein.

– The creation of a termination codon at an inappropriate place is called a “nonsense” mutation.

– β0 thalassemias • Lack of production of β-globin protein due to nonsense mutations giving rise to truncated transcripts that decay rapidly. • Homozygous mutations of splice sites (GU and AG dinucleotides) and nonsensense mutations that introduce premature stop signals at codons 17 (exon 1) and 39 (exon 2) result in β 0 thalassemia.

Question 32

What is a frameshift mutation?

Answer 32

Frameshift mutation

– If one or two nucleotides are either deleted from or added to the coding region of a message sequence, a frame-shift mutation occurs, and the reading frame is altered.

– This can result in a product with a radically different amino acid sequence or a truncated product due to the creation of a termination codon.

– Cystic fibrosis (CF) • Most commonly caused by a deletion of three nucleotides from the coding region of a gene, resulting in the loss of phenylalanine at the 508th position (ΔF508) in the protein encoded by that gene. • Over 70% of patients with CF, the ΔF508 mutation is the cause of the disease. • Mutation prevents normal folding of the CF transmembrane conductance regulator (CFTR) protein. • CFTR normally functions as a chloride channel in epithelial cells, and its loss results in the production of thick, sticky secretions in the lungs and pancreas, leading to lung damage and digestive deficiencies

Question 33

What is Epigenetics?

Answer 33

• Changes that do not affect the DNA sequence of the genome are termed epigenetics
• Affected by the environment
• Alterations in gene expression that are stably inherited
• Two main type of modification – Gene methylation – Direct DNA methylation. – Histone modification by acetylation and deacetylation

Question 34

Describe the process of Gene Methylation in Epigenetics.

Answer 34

Gene methylation – Direct DNA methylation

– The major site of DNA methylation in mammals is on a cytosine base in DNA— especially the 5′ cytosine adjacent to a guanosine base (5′-CG-3′)

– 5′-CG-3′ residues tend to cluster in the promoter regions of genes.

– Important for tissue-specific genes expression.

– Methylation of 5′-CG-3′ residues in DNA is thought to cause steric hindrance to the binding of proteins (transcription factors) that influences gene expression.

– The methylation of a gene is called imprinting. Imprinting is when silencing of genes on chromosomes is a result of gene methylation. These genes are said to be “imprinted” or have the ability to be turned on or off depending on which parent contributed the gene that been silenced

– An example of this imprinting is the Fragile X syndrome (FXS) • Caused by a mutation of the fragile X mental retardation I (FMRI) gene on the long arm of the X chromosome • FMRI normally has 5 to 55 CGG trinucleotide repeats and can potentially expand during meiosis in oocytes. • “Full mutation” is characterized by >200 CGG trinucleotide repeats, which causes FMRI hypermethylation. DNA methylation inactivates FMRI preventing transcription and production of fragile X mental retardation protein. • Symptoms include mild-to-moderate intellectual disability, problems with social interactions and delayed speech, and physical feature that include long and narrow face, large ears, flexible fingers, and large testicles

Question 35

Describe histone modification by acetylation and deacetylation in Epigenetics.

Answer 35

Histone modification by acetylation and deacetylation

– Making DNA more or less accessible to transcription factors

– Lysine residue acetylation weakens the DNA-histone interactions and makes the DNA more accessible to factors needed for transcription .

– Histone acetylation catalyzed by histone acetyltransferases or HATs -> associated with transcriptional activation.

– Histone deacetylation catalyzed by histone deacetylase or HDAC -> associated with gene silencing

Question 36

Describe epigenetics in cancer.

Answer 36

Epigenetics in Cancer

– Epigenetic contribute to cancer etiology

– One major cause of cellular transformation is silencing of tumor suppressor genes • Example is p16 (cell cycle inhibitor/tumor suppressor gene) is epigenetically silenced in many tumors.

– The cancer cell environment its highly dynamic with genomic and metabolic changes that affect gene epigenetic modifications

– Cancer cells may use epigenomic modification for therapy resistance • Silencing of pro-apoptotic genes (death genes) such as Bax and Bak

– Epigenetics can be target for therapy

• Many epigenetic drugs, including two DNA methyltransferase enzyme (DNMT) inhibitors and a deacetylase (HDACs) inhibitor have been approved by the FDA as effective drugs for cancer treatment