Genetics
DNA replication and Chromatin Structure:
● Complementary base pairing is the basis of DNA Replication (self directed replication)
- Proteins bring in appropriate nucleotides
● Performed by DNA Polymerases – carried out
● Although base pairing allows DNA to undergo self replications, DNA polymerases drive
reaction
● DNA polymerases add nucleotides to 3’ OH and require a template to copy and an
existing nucleic acid to attach to and extend (3 characteristics)
- DNA polymerases can correct mistakes using a “proofreading” activity — detects
non bases paired nucleotides
- Error rate: 1 in 10,000 → 600,000 errors in S phase
- Removes incorrect nucleotide and repeats
- Proofreading reduces to 3-6 errors/S- phase (errors are required for evolution)
● In the cell during DNA replication (S phase), DNA synthesis occurs at hundreds of sites
along the chromosomes at sites called replication bubbles
● DNA replication occurs within the replication bubbles at the replication forks
- Replication forks, replication origin, template DNA, newly synthesized DNA
- Slide 5-6
- Bubbles form in DNA double strand and enlarge in both directions, many proteins
are required
● Important proteins required at the replication fork:DNA helicase, Single strand binding,
proteins, DNA Primase (an RNA polymerase), DNA polymerases, RNAse, DNA
topoisomerase
- unwinding the helix via helicase, priming by primase, elongation by DNA
polymerase, and ligation
- Slide 7
● DNA synthesis can be carried out in vitro (in a test tube)– that basis for the Polymerase
chain reaction (PCR) -1984
● Required for the PCR reaction:
- DNA template (can be total human DNA)
- DNA primers (20-30nucleotides) – serve as primer for DNA synthesis and to
pinpoint a specific gene.
- All four nucleotides (G, A, T, C)
- DNA polymerase*
- Heating block, incubator
● Some uses of PCR: Gene cloning, DNA sequencing, criminal justice, mutation detection,
gene expression analysis, infectious disease research, parental testing
● Kary Mullis discovered PCR while on LSD
- Cetus corporation profited over $2 billion, yet Mullis only received $10,000
, ● Telomers: required for chromosome stability/complete replication of the ends of
chromosomes (repeats of nucleotides at the end of chromosomes)
- RNA primer required for DNA polymerase
- RNA primer removed, end not replicated (RNA primer is unstable)
- + end prone to damage
- Telomeres: 6bp repeat added by Telomerase
- Telomere repeat
- TAAGGG –> TAAGGG.. X100
- Bound by telomere binding protein
● As normal cells ‘age’, telomerase activity decreases and telomeres shorten (hayflick
limit)
● Cancer cells (immortal) – high telomerase activity and stable telomeres
● Question: Is shortened telomeres a cause of reduced cell division or a consequence of
reduced division?
- Determined to be a cause how?:
- cloned gene for telomerase > express in normal cells > these cells expressing high
telomerase/stable telomeres are immortalized
● In the nucleus, the DNA is highly organized and compacted by proteins into chromatin
the basic unit of compaction is the nucleosome
● Chromatin is made up of DNA + histone proteins
● Nucleosome: core subunit of chromatin
- Creates a coil with 6 nucleosomes/turns
● Consist of 146bp DNA wrapped around a histone octamer
- Histone octamer: 2x4= 8 histones (H2A, H2B, H3, H4)
● 5 different small histone proteins: H1 (1 copy), H2A (2 copies), H2B (2 copies), H3 (2
copies), H4 (2 copies)
- 80-100 bp linker DNA
● DNA is extensively compacted into chromatin
● Chromosomal DNA and its associated proteins are organized as a cylindrical 30nm fiber
● A loosened fiber shows a “bead on a string” organization
- The string is the DNA molecule, each “bead” is the nucleosome
● A nucleosome consists of part of a DNA molecule wrapped around a core of histone
proteins
● When a chromosome is at its most condensed, DNA is packed into tightly coiled coils
- Organized into loops
The Flow of Genetic Information:
● Central Dogma: DNA→ RNA → Protein
● DNA stores genetic information in the form of nucleotide sequences
● This information ultimately determines:
- The structure (shape) of proteins
, - The function of proteins
● DNA sequence (nucleotides) → determines amino acid sequence
● Amino acid sequence → determines → protein structure and function
● DNA is read in groups of 3 nucleotides= codons
● Each codon codes for one amino acid
● Example: Insulin
- DNA sequence: GGG ATC ATG ACC CCA GTT
- Codons correspond to amino acids: Met – Thr – Pro – Val
- Each codon= specific instruction during protein synthesis
● DNA is located in the nucleus
● Contains genes:
- Genes= stored instructions for making proteins
- Acts like a biological database
● Cells can produce hundreds of thousands of proteins
● The cell follows a step by step process:
1. Cell needs a specific function (example: insulin)
2. Locates the gene in DNA
3. Makes a copy of the gene → mRNA (transcription)
4. mRNA leaves nucleus → goes to ribosome
5. Ribosome uses mRNA to build protein (translation)
6. Protein performs its function
7. mRNA is eventually destroyed
● Transcription (DNA → RNA)
- Occurs in the nucleus
- DNA unwinds
- Enzyme: RNA polymerase
- Creates a complementary mRNA strand
● Translation (RNA → protein)
- Occurs in cytoplasm at ribosomes
- mRNA is read in codons
- tRNA matches (delivers) codons using anticodons (complementary sequence)
Building the Protein:
● Ribosome links amino acids together
● Forms a polypeptide chain
● Chain folds into a functional protein
- Example.) Met – Thr – Pro – Lys – Gly – Pro
● DNA strands
- Coding strand: matches mRNA sequence (except T → U)
- Template strand: used by RNA polymerase to build mRNA
● Movement within the cell
DNA replication and Chromatin Structure:
● Complementary base pairing is the basis of DNA Replication (self directed replication)
- Proteins bring in appropriate nucleotides
● Performed by DNA Polymerases – carried out
● Although base pairing allows DNA to undergo self replications, DNA polymerases drive
reaction
● DNA polymerases add nucleotides to 3’ OH and require a template to copy and an
existing nucleic acid to attach to and extend (3 characteristics)
- DNA polymerases can correct mistakes using a “proofreading” activity — detects
non bases paired nucleotides
- Error rate: 1 in 10,000 → 600,000 errors in S phase
- Removes incorrect nucleotide and repeats
- Proofreading reduces to 3-6 errors/S- phase (errors are required for evolution)
● In the cell during DNA replication (S phase), DNA synthesis occurs at hundreds of sites
along the chromosomes at sites called replication bubbles
● DNA replication occurs within the replication bubbles at the replication forks
- Replication forks, replication origin, template DNA, newly synthesized DNA
- Slide 5-6
- Bubbles form in DNA double strand and enlarge in both directions, many proteins
are required
● Important proteins required at the replication fork:DNA helicase, Single strand binding,
proteins, DNA Primase (an RNA polymerase), DNA polymerases, RNAse, DNA
topoisomerase
- unwinding the helix via helicase, priming by primase, elongation by DNA
polymerase, and ligation
- Slide 7
● DNA synthesis can be carried out in vitro (in a test tube)– that basis for the Polymerase
chain reaction (PCR) -1984
● Required for the PCR reaction:
- DNA template (can be total human DNA)
- DNA primers (20-30nucleotides) – serve as primer for DNA synthesis and to
pinpoint a specific gene.
- All four nucleotides (G, A, T, C)
- DNA polymerase*
- Heating block, incubator
● Some uses of PCR: Gene cloning, DNA sequencing, criminal justice, mutation detection,
gene expression analysis, infectious disease research, parental testing
● Kary Mullis discovered PCR while on LSD
- Cetus corporation profited over $2 billion, yet Mullis only received $10,000
, ● Telomers: required for chromosome stability/complete replication of the ends of
chromosomes (repeats of nucleotides at the end of chromosomes)
- RNA primer required for DNA polymerase
- RNA primer removed, end not replicated (RNA primer is unstable)
- + end prone to damage
- Telomeres: 6bp repeat added by Telomerase
- Telomere repeat
- TAAGGG –> TAAGGG.. X100
- Bound by telomere binding protein
● As normal cells ‘age’, telomerase activity decreases and telomeres shorten (hayflick
limit)
● Cancer cells (immortal) – high telomerase activity and stable telomeres
● Question: Is shortened telomeres a cause of reduced cell division or a consequence of
reduced division?
- Determined to be a cause how?:
- cloned gene for telomerase > express in normal cells > these cells expressing high
telomerase/stable telomeres are immortalized
● In the nucleus, the DNA is highly organized and compacted by proteins into chromatin
the basic unit of compaction is the nucleosome
● Chromatin is made up of DNA + histone proteins
● Nucleosome: core subunit of chromatin
- Creates a coil with 6 nucleosomes/turns
● Consist of 146bp DNA wrapped around a histone octamer
- Histone octamer: 2x4= 8 histones (H2A, H2B, H3, H4)
● 5 different small histone proteins: H1 (1 copy), H2A (2 copies), H2B (2 copies), H3 (2
copies), H4 (2 copies)
- 80-100 bp linker DNA
● DNA is extensively compacted into chromatin
● Chromosomal DNA and its associated proteins are organized as a cylindrical 30nm fiber
● A loosened fiber shows a “bead on a string” organization
- The string is the DNA molecule, each “bead” is the nucleosome
● A nucleosome consists of part of a DNA molecule wrapped around a core of histone
proteins
● When a chromosome is at its most condensed, DNA is packed into tightly coiled coils
- Organized into loops
The Flow of Genetic Information:
● Central Dogma: DNA→ RNA → Protein
● DNA stores genetic information in the form of nucleotide sequences
● This information ultimately determines:
- The structure (shape) of proteins
, - The function of proteins
● DNA sequence (nucleotides) → determines amino acid sequence
● Amino acid sequence → determines → protein structure and function
● DNA is read in groups of 3 nucleotides= codons
● Each codon codes for one amino acid
● Example: Insulin
- DNA sequence: GGG ATC ATG ACC CCA GTT
- Codons correspond to amino acids: Met – Thr – Pro – Val
- Each codon= specific instruction during protein synthesis
● DNA is located in the nucleus
● Contains genes:
- Genes= stored instructions for making proteins
- Acts like a biological database
● Cells can produce hundreds of thousands of proteins
● The cell follows a step by step process:
1. Cell needs a specific function (example: insulin)
2. Locates the gene in DNA
3. Makes a copy of the gene → mRNA (transcription)
4. mRNA leaves nucleus → goes to ribosome
5. Ribosome uses mRNA to build protein (translation)
6. Protein performs its function
7. mRNA is eventually destroyed
● Transcription (DNA → RNA)
- Occurs in the nucleus
- DNA unwinds
- Enzyme: RNA polymerase
- Creates a complementary mRNA strand
● Translation (RNA → protein)
- Occurs in cytoplasm at ribosomes
- mRNA is read in codons
- tRNA matches (delivers) codons using anticodons (complementary sequence)
Building the Protein:
● Ribosome links amino acids together
● Forms a polypeptide chain
● Chain folds into a functional protein
- Example.) Met – Thr – Pro – Lys – Gly – Pro
● DNA strands
- Coding strand: matches mRNA sequence (except T → U)
- Template strand: used by RNA polymerase to build mRNA
● Movement within the cell
