1
Q
Functions of DNA
A
- Stores information
- Replicates faithfully (preservation of information)
- Has ability to mutate (variability of information)
2
Q
DNA stores information
A
- Molecular biology definition of a gene: it is the entire DNA sequence necessary for production of a functional protein or RNA
- the information is carried in the (by the) sequence of bases
3
Q
What type of information does the DNA carry?
A
- Coding for proteins and different RNAs (rRNA, tRNA, small regulatory RNAs)
- Regulatory signals = binding sites
4
Q
central dogma of molecular biology
A
DNA –> transcription –> RNA –> translation –> Protein
5
Q
DNA replicated faithfully
A
- two strands of a parental DNA separate and each serves as a template for synthesis of a new daughter strand by complementary base pairing
- one strand predicts the sequence of the other strand → replication is semiconservative
6
Q
Semiconservative DNA
A
- Semiconservative replication of DNA; two strands of a parental DNA separate and each serves as a template for synthesis of a new daughter strand by complementary base pairing.
- OUTCOME: one strand predicts the sequence of the other strand – information is preserved.
- Implications:
What happens if there is a mutation in ONE of the parental strands?
7
Q
DNA mutates
A
- mutations in coding sequences → possible alteration in protein product
- concept of colinearity of genes and proteins
- mutations could happen in regulatory sequences. Possible consequences?
- formation of new alleles
- altered product (= protein or RNA)
- no product (knock out)
- altered regulation of product expression
- (if mutation in regulatory sequence)
8
Q
nucleic acid bases
A
Purines:
- adenine
- Guanine
Pyrimidines:
- cytosine
- thymine
- uracil
9
Q
sugars
A
Deoxyribose (DNA)
- 2’ does not have oxygen
Ribose (RNA)
- 2’ has an OH group
10
Q
nucleic acid structure
A
Nucleoside
- sugar and base
Nucleotides
- Nucleoside monophosphate
- sugar, base, one phosphate
- Nucleoside diphosphate
- sugar, base, two phosphate
- Nucleoside triphosphate
- sugar, base, three phosphate
11
Q
polynucleotide chain
A
- phosphate group attached to 3’ end of sugar (phosphodiester linkage)
- read 5’ - 3’
- phosphate bound to 5’ of next sugar
12
Q
B- DNA
A
- major groove and minor groove
- one turn is 3.4 nm or 10.5 base pairs
13
Q
major and minor groove
A
- Binding sites for different (regulatory) factors
- Each factor recognizes specific nucleotide sequence on DNA
- Each nucleotide sequence “exposes” specific - unique - distribution of acceptors and donors
14
Q
forces that help form the DNA double helix
A
- Rigid phosphate backbone
- overall negative charge to the molecule - Stacking interactions
- Van der Waals interactions between bases (weak, but many) - Hydrophobic interactions
- highly negative phosphate backbone “outside” vs. nonpolar (hydrophobic) bases “inside” - Ionic interactions
- salts (+ve ions) stabilize phosphate backbone (DNA shielding) - Hydrogen bonding
- is responsible for complementary base pairing but is not the most energetically significant component
15
Q
DNA forms
A
A-DNA
- right handed
- major groove is deep and narrow
- minor groove is shallow and broad
- 11 bases per turn
- low humidity, high salt condition
B-DNA
- right handed
- major groove is moderate depth and wide
- minor groove is moderate depth and narrow
- 10.5 bases per turn
- high humidity, low salt condition
Z-DNA
- left handed
- major groove is very shallow, almost non-existent
- minor groove is very deep and narrow
- 12 bases per turn
- in presence of methylated cytosine: high humidity, low salt
16
Q
triple helix DNA
A
- Formed when purines make up one strand and pyrimidines the other, then a third strand can be accommodated
- In test tube, but also likely in vivo during DNA recombination or repair
- gene therapy possibilities
17
Q
important characteristics of B-DNA
A
- average 10 base pairs per turn
- 0.34 nm rise per base pair
- 2nm diameter
18
Q
factors that denature DNA
A
- Heat
- Low ionic strength
- promotes repulsion between negative phosphate back-bones (low salt)
- High pH:
- “stripping” of H+ shared between electronegative centers (NaOH)
- Agents that influence H-bonds
- competition:
- have functional groups that can form H-bonds with the electronegative centers (NH2- and O=; urea, formamide)
- covalent modifications:
- modify electronegative centers and block the formation of H-bonds (formaldehyde, glyoxal)
- competition:
- Agents that enhance the solubility of hydrophobic substances (organic solvents, temperature, pH,)
19
Q
monitoring DNA denaturation
A
- The progress of denaturation can be monitored by examining the properties of the molecule that change when the strands separate
- Viscosity – rarely used….difficult
- Absorbance (260 nm)-commonly used in the laboratory
20
Q
How does absorption spectrophotometry work?
A
Tm: melting temperature
- temperature at which 50% of the DNA is denatured
Absorbance changes depending on the stacking of purines and pyrimidines:
- In double stranded DNA the bases are stacked and absorbance is lower (hypochromic)
- In denatured single stranded DNA the bases are unstacked and absorbance increases (hyperchromic)
21
Q
Hypochromic Effect
A
stacked bases have low absorbance
22
Q
Hyperchromic Effect
A
un-stacking of bases causes increase in absorbance
23
Q
denaturation and GC content
A
- Tm is a function of the GC content
- More GC : higher Tm needed
- AT regions separate first during denaturation
- The Tm of DNA increases by 0.4oC with every 1% increase in G-C content under normal condition
- Higher salt = higher Tm
24
Q
renaturation
A
- Renaturation (and hybridization) is the recombination of two complementary single stranded DNA
- Dependent on:
- DNA concentration - complementary single strands must “find each other” (number of copies)
- Salt concentration - ionic conditions - mask repulsion forces of phosphate backbone
- Temperature : 20 - 250C below Tm
- Time (reaction time)
- Size of the DNA fragment (length)
- Complexity - simple sequences re-nature faster than complex sequences
- These properties can be used to analyze and classify DNA.
25
CoT analysis
- Rate of renaturation = measure of complexity of DNA/genome
- Re-association kinetics: speed at which a single strand sequence is able to find a complementary sequence and base pair with it.
- Expect: increase in genome size = increase in complexity
- Simple sequences re-nature more quickly than complex sequences
Co = starting concentration (nucleotides per liter)
t = reaction time (seconds)
26
CoT analysis conditions
- Units of complexity are measured in terms of nucleotides
- If a genome (or DNA sequence) is all unique (nonrepeating) in sequence then:
- Complexity = # of nucleotides
- If a genome (or DNA sequence) contains unique sequences and some repetitive sequences then:
- Complexity = # of ‘unique’ nucleotides + total # of nucleotides from one copy of each repetitive sequence
- If two DNA sequences do not have repetitive sequences (their sequences are UNIQUE) and have similar C-G contents, their sizes are proportional to their Cot1/2
27
complexity examples
DNA composed of the repeating copolymer dAT (ATATATATAT) has a complexity of 2
DNA composed of the repeating tetrameric sequence (ATGC)n has a complexity of 4
A DNA composed of 105 non-repeating nucleotide pairs in length has a complexity of 10^5
A DNA composed of 105 non-repeating nucleotide pairs, plus 100 copies of dAT, 50 copies of (ATGC) in length has a complexity of 10^5+2+4
28
How is a CoT analysis carried out
Control and Unknown DNA --> sheared into small pieces (200bp) --> denatured using heat --> allowed to cool slowly (re-anneal) --> sub samples removed, ds and ss DNA measured with absorbance at 260nm --> data points plotted as a proportion of ssDNA out of the total DNA
29
circular DNA
- Circular genome is composed of two strands of DNA that form a closed structure without free ends = “double circle”
- Prokaryotic genomic DNAs, plasmids and many viral DNAs are circular
- Chloroplast and Mitochondria also have circular genomes
- Endosymbiotic theory.
30
denaturation of circular DNA
- Circular DNA can also be denatured like linear DNA
- However, two strands cannot unwind and separate like linear DNA
- In vivo, nicking occurs naturally during DNA replication
- Can be induced experimentally by using an enzyme
31
both circular and linear DNA
- Primary structure of DNA: sugar-phosphate “chain” with purine and pyrimidine bases as side chain(s)
- Secondary structure of DNA double helical structure (hydrogen bonding between A-T and G-C; stacking interactions; phosphate backbone “outside”)
- Tertiary or higher order structure double stranded DNA (both circular AND linear) makes complexes with proteins – supercoil
- Supercoiling = coiling of a coil.
32
supercoils
- reduce stress on DNA by twisting/untwisting the double helix
- Topological isomers- DNA differing only in their states of supercoiling A circular DNA without any superhelical turn is known as relaxed molecule.
- Important for packing of DNA – circular or linear one way of making DNA more “condensed”
- DNA helix becomes topographically linearized (locally uncoiled) during replication and transcription
- Base paring is interrupted
- DNA molecule exhibits supercoiling
33
supercoiling
If we “open” (unwind) a region of DNA:
- If one end is “free”: just “untwist”.
- If both ends are “fixed” = situation in circular DNA molecules AND locally in long linear DNA molecules (bound by chromatin scaffold)
- Strain is released by writhing into superhelical turns (supercoils)
- One DNA supercoil forms in the double helix for every 10 bp opened (for B DNA).
34
positive supercoils
- Protein “opens” dsDNA two strands in front of the opening become wrapped around each other more than once every 10bp – overwinding (turns are “shorter”- fewer bases/“new” turn)
- Consequence: formation of positive supercoils in front of the opening; occurs when right-handed B-DNA is twisted really tightly about its axis, the double helix begins to distort and knot (stress!) into positive left-handed supercoils.
35
negative supercoils
- Protein “opens” dsDNA two DNA strands behind the opening become wrapped around each other less than once every 10bp – underwinding (turns are “longer”- more bases/ “new” turn)
- Consequence: formation of negative supercoils behind the opening; “loosing” the tension and causing the B-DNA helix to start un-twisting/un-winding, thereby increasing stress leading to negative right-handed supercoils.
36
Twisting #: T
- the crossing of one strand of dsDNA over the other. It measures how tightly the helix is wound.
- For a 2,000 bp DNA duplex (we assume it’s a “normal” - B DNA)
- T = 200 (2,000 bp divided by 10bp/turn = 200 turns)
T= Total # of base pairs/ #base pairs per turn
(T is positive for A and B DNA, negative for Z DNA)
37
Writhing #: W
- number of superhelical turns; refers to the twisting of the dsDNA axis in space (how many times the duplex DNA crosses over itself)
- Relaxed dsDNA: W=0
- Negative supercoils: W is negative
- Positive supercoils: W is positive
38
Linking #: L
- total # of times one strand of closed molecule of dsDNA encircles the other strand (integer). It reflects both the twisting (T) of the native DNA helix and the presence of any supercoiling (W)
- L can only be changed by breaking one or both strands of the DNA, winding them tighter or looser, and rejoining the ends = change W.
- L is a constant in unbroken duplex DNA, so any change in T must be accompanied by an equal and opposite change in supercoiling
L=T+W
39
topoisomerases
- Most cell DNAs are negatively supercoiled
- Negative supercoils store energy – energy of negative supercoils can be converted into untwisting (unwinding) of double helix
- DNA OVERWOUND - positive supercoiling: reduced chance for DNA-protein interaction
- DNA UNDERWOUND - negative supercoils negative supercoils store energy that could help strand separation - untwisting favored (important for replication and transcription)
- Topoisomerases are enzymes that recognize and regulate supercoiling and play an important role in replication and transcription
40
prokaryotic
- Pos and Neg supercoils essential in prokaryotes – studies with mutants
- Topoisomerase I: nicking-closing enzyme, makes transient cuts in one strand – relaxes negative supercoiling in prokaryotes. Changes L # in steps of 1
- Topoisomerase II – relaxes positive supercoiling (uses ATP). Makes double-stranded cut, pass a duplex DNA through it and re-seals the cut. Changes L # in steps of 2.
- Gyrase (one of bacterial Topo II): introduces negative supercoils
- Reverse gyrase - discovered in the hyperthermophilic archaebacterium, Sulfolobus. Topo I generating positive supercoils (requires ATP)
- stabilizing the genome structure at high temperature (genetic knock-out experiments: reverse gyrase mutant is viable but shows significant growth defects at high temperature)
- protecting the DNA strand breakage promoted by exposing DNA to high temperature
41
Different types of RNA
- mRNA - messenger RNA, specifies order of amino acids during protein synthesis
- tRNA - transfer RNA, during translation mRNA information is interpreted by tRNA
- rRNA – ribosomal RNA, combined with proteins aids tRNA in translation
- Small RNAs – variety of regulatory functions
- RNAs with enzymatic functions – ribozymes (in splicing, and peptide bond formation during protein synthesis)
42
Structure of RNA
- 2’ OH group prevents formation of B-helix: A-helix is formed
* RNA, can be single or double stranded, linear or circular.
* Unlike DNA, RNA can exhibit different conformations
* Different conformations (secondary and tertiary structure formation) permit different RNAs to carry out a variety of specific functions in the cell
43
secondary structures in RNA
* RNA molecules frequently fold back on themselves to form base-paired segments between short stretches of complementary sequences
* G:U = additional, non-Watson & Crick base pairing possible in RNA → enhances potential for self-complementarity in RNA
* Secondary structures: areas of regular helices and discontinuous helices with stem-loops or hairpins
44
tertiary structures in RNA
* formed through interactions of secondary structures: lack of constraint by long-range regular helices means RNA has high degree of rotational freedom in backbone of its non-base-paired regions → capable of folding into complex tertiary structures
* also, formation of unconventional triple base pairing is possible
* pseudoknots can form due to base-pairing between sequences that are not adjacent
* Telomerase RNA has “pseudoknots”
45
bacterial chromosome
- Genome forms a compact structure called the nucleoid- mixture of supercoiled and relaxed regions
- DNA organized in 50-100 loops (domains)
- Circular molecule is compacted by association with:
* Polyamines (spermine and spermidine, +ve charge),
* HU proteins (small, basic, +ve, dimeric) and H-NS (monomeric, neutral) DNA binding proteins
* Supercoiling
46
restrained supercoiling
path is supercoiled around protein but creates no tension
47
unrestrained supercoiling
path is supercoiled in space and creates tension
48
eukaryotes
- The average size of chromosome is 150Mbp=150x103 kbp=150x106bp
- Length of this DNA would be 150 x 106 bp x 0.34 nm =51x106 nm=51mm
- There are 46 chromosomes in human somatic cell
= 51 mm x 46 = 2346 mm Þ approx. 2.5 m of DNA/ human cell
- The average size of eukaryotic cell is anywhere from 10 to 100 μm (and DNA is in the nucleus, which is even smaller!!!!)
- Eu cell has to solve even more challenging problem then Pro cell:
- How to pack 2.5 m (of 2 nm thick) thread into a ball 10 μm in diameter?!?!
49
eukaryotic chromatin
- Eukaryotic genomes organized to form linear chromosomes
- Each chromosome contains single, linear DNA molecule
- Nucleoprotein material of eukaryotic chromosome is chromatin
- Individual chromosomes can be separated by pulse-field gel electrophoresis
* electric field repeatedly alternated
* chromosomes migrate as distinct bands on gel
- by dark field electron microscopy, fiber structure of a chromosome resembles “beads on a string”
50
chromatin organization
- “Beads on a string”:
- The fundamental unit of organization of the chromatin fiber is nucleosome
- Each nucleosome contains a core particle of histone proteins which are wrapped by DNA.
- Also: nonhistone proteins are involved and important for chromatin organization
51
Histones
- Present in all eukaryotic nuclei
- Small proteins rich in lysine and arginine (at normal pH their “extra” amino groups become NH3+)
– basic proteins
- Interact with DNA through
electrostatic interactions
- Five major subunits:
* H1(Linker), H2A, H2B, H3 and H4
* H2A, H2B, H3 and H4 form a complex of 8 proteins (octet)
52
nucleosomes
- Nucleosome = octet of histones and wrapped DNA
- DNA (147 bp) wraps around octet approximately 1.65 times
- H1 associates with DNA and octet in linker region – binds two distinct regions of the DNA duplex
53
chromatin organization: EM
10 nm fibre
- likely a consequence of unfolding during extraction in vitro
- H1 is not required
30 nm fibre
- basic constituent of interphase chromatin, mitotic chromosomes
- “tighter packing” requires H1
54
fibres
- Core histones alone: ~ 4-fold
compression
- Core histones + H1: 25- to 100-fold compression
- Nucleosome is 10 - 11 nm in diameter
- 10 nm fiber is “re-packed” into a 30 nm
diameter fiber
- higher salt concentration during isolation - different forms (10 nm nucleosomes - beads to more condensed 30 nm fibers).
- Recent electron microscopic studies- dynamic structure
55
Chromosome structure
- Euchromatin consists of transcriptionally
active DNA, susceptible to DNase digestion
- Heterochromatin less susceptible to DNase digestion and transcriptionally inactive
a) constitutive heterochromatin - highly condensed
inactive chromatin; consists of repetitive DNA, very few genes
- Centromere – specific sequences; attachment point for sister chromatids and spindle fibers (constitutive heterochromatin)
- Telomere - end of chromosome (constitutive heterochromatin)
b) facultative heterochromatin - not active in particular tissue. Forms under specific circumstances and/or certain tissues to silence gene expression:
- X-chromosome inactivation (Barr body formation)
- Imprinting (heterologous allele “silencing”)
56
chromatin elements and centromeres
- Chromatin elements (elements = specific nucleotide sequences, DNA regions!)
- Locus control regions – shared control regions (usually upstream from gene clusters) – control chromatin condensation (role in extrusion?)
- Matrix and scaffold associated regions – mostly AT-rich DNA which anchors to the nuclear matrix
- Insulators – regulatory domains in DNA - define domains of gene expression, possibly recognized in extrusion process?
- Centromere = chromosome region which contains the site of attachment for spindle fibres. Kinetochore = Centromere + proteins (connect to fibres)
- In situ hybridization of metaphase chromosomes shows satellite DNA at centromeres (highly repetitive sequences)
- kinetochore = centromere with proteins
- spindle fibres attaching at centrioles
57
telomeres
- Specialized regions of DNA at the ends of chromosomes.
- Repetitive sequences as well
- Protect chromosomes from shortening during replication and from degradation (by “looping” of the 3’ overhang)
58
nonhistone proteins
* matrix attachment regions (MARs) or scaffold attachment regions (SARs) DNA elements bound by scaffolding proteinaceous structures
* suggested: this binding is required for replication and transcription
* MARs A:T rich (~70%), but no consensus sequence
* may include cis-acting sites that regulate transcription
* usually recognition site for topoisomerase II (role in packing)
* structural maintenance of chromosome (SMC) proteins - responsible for scaffolding (metaphase structures)
* DNA replication proteins
* transcriptional factors, chaperone proteins etc
59
histone proteins
- Highly conserved proteins especially H4
- Genes repeated 10-20 times in mice, 100 times in Drosophila
- Some of gene copies are quite different
- H1 – least conserved, most variations (six subspecies in mice)
- Fish, amphibians, reptiles and birds have another lysine – rich histone – an extreme variant of H1, called H5
- Specialized: H3 variant specific for centromeres – CenH3
60
Nucleosome disassembly and reformation
- Replication of DNA requires partial disassembly of the nucleosome
- Newly replicated DNA immediately packed: first bind H3-H4 tetramer and then two H2A-H2B dimers, H1 is last
- H1 induces tighter DNA wrapping around the nucleosome
- More DNA (due to replication) - newly synthesized histones are required
- Old and new histones present on both daughter chromosomes
- Chaperone proteins – negatively charged proteins, assist assembly of histones (escort dimers to the replicating target DNA)
- Replicating DNA is absolutely necessary for nucleosome assembly
61
histone tails
- Histones have N-terminal tails extending out from nucleosome –
several +ve lysines (basic amino acids – “extra” amino group)
- Protruding tails serve as the “grooves of the screw” – direct the DNA wrapping
- Tails are necessary for the formation and the stabilization of the 30 nm fiber through interaction with adjacent nucleosomes
62
Transcriptionally Active Genes and Chromatin Condensation
- Eukaryotic transcription and replication occur in the context of chromatin:
- Modification of chromatin necessary for the replication to start and for modulation of gene expression
- Transient modifications of amino acids in histones’ tails
* Acetylation of histone tail (lysine) gen. associated with active gene expression
* Ubiquitinylation of histone tails (lysine) “mono-” = non-destructive modifications
* Methylation of histone tail (arginine and lysine) – may be associated with active or inactive genes (depends on other factors)
* Phosphorylation (serine) – generally associated with active gene (not completely clear as histones are phosphorylated during mitosis)
63
the histone code hypothesis
- Serial modifications of histones’ tails are “landmarks” for proteins which “read” chromatin (domains recognize modified tails)
- Creates “open” chromatin necessary for transcription, replication, repair, and recombination
64
Acetylation and Deacetylation of histone tails
- Enzymes: HISTONE ACETYLASES and DEACETYLASES
- Acetylated form is NEGATIVE (+H atom replaced by –acetyl group)
* Affects chromatin’s condensation (open)
* Necessary for activation of transcription
- Nucleosome free regions – contain actively transcribed DNA (gene expression in progress) – DNase-sensitive regions
- Methylation and Acetylation of lysine
and Phosphorylation of serine reduces overall positive charge of protein
65
DNase I test
- Isolate nuclei, treat with diff. conc. of DNase
- Separate protein, DNA
- RE digestion, Gel electrophoresis, Southerns (globin & vitellogenin), two possibilities:
1. No signal - gene expression was in progress, DNA was free of histones and available for digestion by DNase
2. Signal - no gene expression...
66
prokaryote vs eukaryote
Prokaryotes
- No membrane-bound nucleus
- Single, circular chromosome
- NO membrane-bound organelles
- Single cell organisms
Eukaryotes
- All have a membrane- bound nucleus
- linear chromosomes # varies by species
- Have membrane-bound organelles (organelle DNA)
- Can be single cells or multi-cellular organisms
67
cells go through division for
MITOSIS
- Tissue Growth – Larger bones, bigger muscles
- Repair of damaged tissues – Broken bones, muscle tears, renewed skin cells, broken blood vessels
MEIOSIS
- Reproduction – requires specialized cells = GERM CELLS specialized division to produce sex cells = GAMETES
68
cell division and the cell cycle
- Orderly set of events that take place between the formation of a new cell and the division of that “parent” cell into 2 new “daughter” cells is called cell cycle
- Two Main Stages
* Interphase: normal cell functions and preparation for mitosis
* Mitosis and cytokinesis: nuclear division and final division/separation of the “parent” cell cytoplasm into two “daughter” cells
69
interphase
* Most of a cell’s life is spent in interphase.
* G1, S, G2
* Normal cell functions are carried out in interphase (protein synthesis is constantly going on; proteins needed for...).
* Some cells never leave interphase – stop dividing (G0 – pre S)
70
phases of cell cycle
G1: cell grows, carries out normal metabolism, organelles duplicated
S: DNA replication, chromosome duplication (S stands for synthesis)
G2: cell grows, prepares for mitosis
M: mitosis: prophase, prometaphase, metaphase, anaphase, telophase; cytokinesis at the end
G0: cells that have stopped dividing (may be temporary, permanent)
71
mitosis
- Mitosis is nuclear division - the nucleus and its contents are divided.
- 4 phases:
* Prophase
* Metaphase
* Anaphase
* Telophase
72
check points in cell cycle
- The cell cycle is very tightly controlled;
control system similar to a clock
- The frequency of cell division varies with the type of cell
- These differences result from regulation at the molecular level
- At specific times during cell cycle, specific proteins (cyclins and cyclin-dependent kinases, whose activity fluctuates during the cell cycle) STOP the cell cycle to make an assessment.
- Those stops are called CHECKPOINTS.
- If everything seems in order: the cell cycle “machinery” continues to the next stage.
- If something is wrong: the “machinery” waits until the error is corrected.
- If the error cannot be corrected: the cell will undergo apoptosis (programmed cell death).
73
what is the purpose of check points
- Energy and resources are saved - no good reason to make a copy of a damaged cell
- Internal control – through internal signals; for example kinetochores not attached to spindle microtubules send a molecular signal that delays anaphase – could be corrected
- External control is enabled as well (eg. growth factors; density dependent inhibition etc.)
74
more on checkpoints
- occur throughout cell cycle; assess & control readiness of cell to proceed
- each checkpoint represents control loop - makes initiation of one event dependent on successful completion of an earlier event
*division: external stimuli (e.g., nutrients), cell mass influence, properly completed mitosis
*DNA integrity at the beginning of replication is critical
*success of replication: completion of DNA replication is critical
*mitosis: paired kinetochores etc.
75
checkpoints can be triggered
- Successful cell division requires coordination of 2 cycles:
- DNA must replicate once & only once (licensing factor)
* must not try to divide cell until DNA replication is complete (check point at mitosis)
- Mass of cell must double to provide material for daughter cells
* must not start to replicate unless mass will be sufficient
* cell mass affects ability to move through START
- Several events (internal and external signals) can trigger checkpoints → cycle arrested
e.g., check for DNA damage at every stage
76
meiosis
- How to preserve the information while making it VARIABLE at the same time?
– variability is very important for survival if any conditions change
- Resolution of the paradox = Sexual Reproduction USING Meiosis.
- Sexual Reproduction:
1. production of special reproductive cells, or gametes (1n)
2. fusion of TWO gametes (fertilization) – zygote (2n)
- Gametes produced in specialized tissues (germ line) through reductional cell division (2n --> n) = MEIOSIS
77
reduction of chromosome number
- only one round of DNA replication (= one duplication of genetic material), but two meioitic divisions
1. homologous chromosomes, containing duplicated DNA, separate (2n --> n)
2. sister chromatids separate (n --> n) This creates the potential for TWO types of variability:
78
Variability 1:
- the Independent Assortment of Homologous Chromosomes in Meiosis
- The number of possible new combinations
of chromosomes for a species is 2n
n = haploid number of chromosomes for the species
79
variability 2:
- crossing over during meiosis
80
mitosis vs meiosis
MITOSIS
- An equational division that separates sister chromatids Equational division – maintains the ploidy/ of chromosomes in the cell.
- One division per cell cycle;
one cytoplasmic division per equational chromosomal division
- Homologous chromosomes do not synapse; no chiasmata
- No genetic exchange between homologous chromosomes
- Two daughter cells per cycle
- Genetic content of mitotic daughter cells is identical to mother cell’s genetic content
- Chromosome # of daughter cells is same as mother cell
- Daughter cells are usually capable of undergoing additional mitotic or meiotic division
- Normally occurs in almost all somatic cells
- Begins at the zygote stage and continues through the life of organism
MEIOSIS
- The first stage (meiosis I) is a reductional division that separates homologous chromosomes; Sister chromatids separated in an equational division during the second stage (meiosis II)
- Two divisions per cell cycle; One cytoplasmic division follows reductional chromosomal division (from 2n to n) Second cytoplasmic division follows equational chromosomal division
- Homologous chromosomes synapse and form chiasmata
- Genetic exchange between homologous chromosomes occurs = variability!
- Four daughter cells / cycle = GAMETES
- Genetic content of meiotic daughter cells is different from each other’s and from mother cell’s genetic content
- Chromosome # of daughter cells is half that of the mother cell
- Daughter cells cannot undergo additional meiotic division although they may undergo subsequent mitotic divisions. (gametophyte vs. sporophyte forms)
- Occurs only in specialized cells of the germ line
- Occurs only after higher organisms have begun to mature (in majority of higher organisms)
81
DNA replication
- Replication is semiconservative
- Efficient – it always starts at a defined sequence of base pairs
- Replication origin: recognized DNA sequence necessary and sufficient for beginning of replication
* bacterial genomes and plasmids often have only one
* more complicated in eukaryotic genomes – multiple origins
- Entire region of DNA replicated from one origin is a REPLICON (= a segment of DNA which replicates as a single unit)
- Bidirectional from the origin in majority of organisms:
* Replication bubble
* Replication forks
* Leading and lagging strands
82
bidirectional confirmation
- Cultured mammalian cells
- Given a “pulse” of 3H-T, followed by a “chase” of unlabeled T. (“pulse-chase experiment”)
- Helps identify where T is being incorporated in close to “real time”
- cells treated to release DNA and dried on a microscope slide
- Autoradiographic treatment of slides (dipped in photographic emulsion, then developed).
- “Hot” areas (incorporated 3H-T) will expose film, reveal pattern.
- DNA labeled near replication origin will be “hot” (from pulse) and will show decreasing label (from chase) over time – two patterns possible
83
discovery of enzymes involved in DNA replication
- DNA replication first studied in prokaryotes using both
genetic and biochemical approaches:
- Make a mutant population and screen for mutants affected in DNA replication
- Characterize as to whether replication stops quickly or slowly under non-permissive conditions
- Purify enzymes required for replication in vitro
- Assay: Add radiochemical dNTP to an extract containing enzymes necessary for replication*, along with a template DNA and measure incorporation of radioactivity into new DNA (can be precipitated and collected on a filter)
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DNA replication machinery
- Several enzymes and proteins involved in DNA replication (Major enzymes/proteins to know)
- DnaA - initiation
- Single-strand binding
proteins (protection)
- Helicase (DnaB) and Primase (DnaG, RNA Polymerase)
- Clamp loader (DnaC) Sliding clamp (ß clamp)
- DNA polymerase III
- DNA polymerase I & RNase H
- DNA ligase
- Topoisomerase
- Replisome: combination of all the proteins that function at the replication fork, & undertake the synthesis of DNA
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why is replication so complicated
- Three elementary problems in copying of DNA by DNA polymerase:
1. DNA polymerase cannot break inter-chain hydrogen bonds at the point of origin – other enzymes/proteins are necessary for replication initiation and strand separation
2. DNA polymerases cannot start chains, only elongate them – it needs “primer” (3’-OH) – made by different enzyme/process Þ oligo-ribonucleotide made by specific RNA polymerase
3. DNA polymerase can add nucleotides only at 3’- OH end: chain always (only) grows in 5’- 3’direction (added at 3’-OH) --> which is problem because we know that:
* DNA is double stranded (ds),
* strands are antiparallel, and
* replication is semiconservative
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the 3'-OH problem
- There are alternate ways to generate 3’ ends; they are used by different organisms
- Specific RNA polymerase
- Synthesize small segment of RNA Happens during “normal” DNA replication in both Pro and Eu Retroviruses can supply preformed RNA
- Nicked DNA
- Happens in rolling circle replication: some phages do this
- Priming nucleotide
- Some viruses can do this; Also happens at the end of eukaryotic replication (telomerase)
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events in growing fork/ replication bubble
- Two strands replicated in different ways:
- Leading strand –continuously synthesized from a single primer on the leading strand template; grows in 5’-3’direction
- Lagging strand – discontinuous synthesis from multiple primers on the lagging strand template in several steps (still 5’-3’)
- Discontinuous strands on lagging = Okazaki fragments
- Coordination of leading and lagging strand elongation is essential for replication
- One replication bubble = two replication forks - one DNA strand will replicate as both leading and lagging strand starting from the origin of replication
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BIOL 308
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