Biochemistry Lecture 3 + 4: Dna Mutations And Replication

Front 1

How many chromosomes in somatic, diploid cells?

Back 1

46 chromosomes (2 each of numbers 1-22 + 2 sex chromosomes)

Front 2

Females vs. males chromosomes

Back 2

Females have 2X's
Males have X and Y
- One from dad and one from mom

Front 3

Compare cells in the body/ makeup of DNA and expression of genes

Back 3

- Every single cell in the body has same cell material/ DNA
- Expression of genes changes

Front 4

Size of DNA

Back 4

48-280 million basepairs

Front 5

What phase is DNA replicated

Back 5

S phase

Front 6

When do daughter sister chromatides become attached to each other?

Back 6

G2, M phase

Front 7

Centromere

Back 7

joining site, site of microtubule attachement during movement of sister chromatids to opposite m=poles o=in mitoses and meioses

Front 8

What is centromeric DNA composed of?

Back 8

AT-rich, short (171 bp) satellite repeats

Front 9

What is a telomere

Back 9

- ends that protect DNAs from shortening during replication

Front 10

Number of genes in humans vs. mouse

Back 10

27,000-mouse
29,000-human

Front 11

Transposon

Back 11

piece of DNA that can jump from one position to another
- create antibiotic resistance in bacteria

Front 12

What percentage is the protein coding region?

Back 12

1.5%

Front 13

Breakdown- genes, transposons, misc

Back 13

Genes- 30%
Transposons- 45%
Misc- 25%

Front 14

What is included in breakdown of transposons?

Back 14

- 21% LINEs
- 13% SINEs
- 8% retrovirus like

Front 15

What is a gene?

Back 15

- entire DNA sequence required for synthesis of useful RNA (rRNA, tRNA, mRNA or other RNA)
- includes promoters, enhancers, UTR's

Front 16

What amount is expressable sequences?

Back 16

29%

Front 17

The protein-encoding, reading frames (without introns) are what percent of genome?

Back 17

1.4%-

Front 18

What is the rest of DNA that is not expressable sequence or protein encoding?

Back 18

Spacer DNA between genes, and half of the spacer DNA is derived from ancient mobile genetic elements, some of which is still jumping form place to place in the genome

Front 19

Introns + Exons in Ovalbumin gene and hemoglobin B subunit

Back 19

Ovalbumin- 7 introns, 7 exons
Hemoglobin- 3 exons, 2 introns
90, 131, 222, 851, 126
Ex, In, Ex, In, Ex

Front 20

4 steps of cell cycle, what is interphase and mitosis?

Back 20

S, G2, M, G1
Interphase- G1, G2, S
Mitosis= PMAT

Front 21

Cell cycle: what process occurs between S and G2?
- What are cohesins

Back 21

replication and cohesion
- Replication occurs from multiple origins of replication; daughter chromatids are linked by cohesins

Front 22

What occurs between G2 and Prophase of mitosis?

Back 22

Condensins add for condensation

Front 23

What leaves after prophase? What happens during this time?

Back 23

Cohesins leave
- Alignment of chromatids

Front 24

What happens in metaphase?

Back 24

DNA condensed

Front 25

What occurs between metaphase and anaphase?

Back 25

separation of sister chromatids

Front 26

What occurs between anaphase and G1?

Back 26

- Condensins leave

Front 27

Trace chromatid --> DNA

Back 27

Two chromatids: 10 coils each
One coil: 30 rosettes
One rosette: 6 loops
One loop: 75,000 bp
30 nm fiber
"Beads on a string" form of chromatin
DNA

Front 28

Mutation definition

Back 28

permanent change in nucleotide sequence

Front 29

Point mutation

Back 29

permanent change of single nucleotide

Front 30

Long term DNA mutation rate

Back 30

Stable DNA mutation rate (mutations in gene pool) is 1% / million years
These mutations are basis of evolutionary process

Front 31

Short term evolution rate
- What happens when somatic cell mutates?

Back 31

- Mutations in somatic cells are more frequent and cause 20% of premature deaths in western hemisphere
- Most cancers are clones of cells with mutated DNA

Front 32

Protecting somatic cell protects who? Protecting germline cell DNA protects...

Back 32

- Somatic protects individual
- Germline protects species
(sperm and oocytes)

Front 33

Types of Mutations (6 types)

Back 33

mis- replication
depurinations
deaminations
alkylations
pyrimidine dimers
other

Front 34

Mis-replication (frequency and cause)

Back 34

- 10^-8 frequency
- DNA damage from environmental factors
- 1 error every 10 billion letters

Front 35

Depurinations- what is it, how, how many

Back 35

- Spontaneous hydrolysis of the beta N-glycosidic bonds
- Heat fluctuations in nucleus
- 5000 / cell/ DAY

Front 36

How do deaminations occur? How many?
What are the products?
C, A, G deaminate to what products?

Back 36

- Spontaneously
- 100 / cell/ DAY
- Unnatural base is product
C --> Uracile
A--> HX hypoxanthine
G --> X (xanthine)

Front 37

Alkylations include what type of mutations? What is the effect?

Back 37

- Methylations (metal group attaches)
- Large adducts such as benzo(a)pyrene from smoke
- Distort DNA

Front 38

Pyrimidine dimers- what happens? How?

Back 38

- UV induced covalent bonds between adjacent pyrimidines on same strand

Front 39

Other types of mutations

Back 39

- Ring opening
- Oxidation
- Cross-linkage
- Single and double strand breaks

Front 40

Point Mutations (3)

Back 40

Transitions
Transversions
Single base insertions/ deletions

Front 41

A. Transitions, give example

Back 41

Purine converted to different purine
A--> G
Pyrimidine converted to different pyrimidine C--> T

Front 42

With transition mutations, what happens after a round of replication to the base pairs?

Back 42

- A-T becomes G-C
- C-G becomes T-A

Front 43

B. Transversions, give example

Back 43

Purine converted to pyrimidine
Pyrimidine converted to purine
A-C, A-T, G-C, G-T

Front 44

When transitions or transversions occur in gene-encoding a protein, what are 3 possible consequences?

Back 44

Neutral- no change in AA
Missense- substitution change of one amino acid
Nonsense- creation of a stop codon leading to truncation of the AA sequence

Front 45

Single Base Insertions/ Deletions: causes, consequences

Back 45

- Caused by DNA polymerase stuttering
- If in protein reading frame, results in codon frameshift- all downstream codons incorrect
- Accidental stop codon crops up in new aberrant reading frame

Front 46

DNA Replication- what is it?

Back 46

- Make or reproduce an exact copy

Front 47

Eukaryote- length of DNA
- Drosophila
- Human

Back 47

Drosophila kB = 165, 000
length = 56,000

Human kB = 2,900,000
Length = 990,000

Front 48

How many origins of replication do stem cells have?

Back 48

20,000 origins of replication

Front 49

Complexity of DNA- packaging

Back 49

All eukaryotic DNAs are folded and packaged into dense, compact structures
- Associated with chromatin

Front 50

What is chromatin?

Back 50

tightly packed DNA-binding proteins which help to organize the package

Front 51

What is relationship between complexity and replication?

Back 51

- Complex structures are impediments to the replication mechanism that requires that two parental strands be totally separated by the end of the process

Front 52

What is the meaning of DNA replicated semi-conservatively?

Back 52

- Each daughter molecule ends up with a parental strand and a nascent strand

Front 53

How is replication semi-discontinuous?

Back 53

One strand is replicated continuously from 5'--> 3'
One strand replicates on 3'--> 5' strand, but discountinuously in segments

Front 54

So 2 characteristics of DNA synthesis

Back 54

- Semidiscontinuous
- Semiconservative

Front 55

Where does replication start?

Back 55

- Origin of replication, or multiple origins in nuclei

Front 56

How is timing of replication of both strands related?

Back 56

Growth of each "replicon" is bidirectional with both strands being replicated simultaneously

Front 57

What enzyme works in replication?

Back 57

Synthesis is carried out by DNA polymerases

Front 58

What direction do DNA polymerases synthesize?

Back 58

5' to 3' direction
- READ TEMPLATE IN 3' to 5' direction

Front 59

What is proofreading by DNA polymerase?
- What 2 types of polymerases lack proofreading?

Back 59

3' to 5' exonuclease activity is called proofreading
- Polymerases alpha and beta are exceptions that lack proofreading

Front 60

What types of polymerase have 5 to 3' exonuclease activity that functions in repair? What other activity are they involved in?

Back 60

- Prokaryotic DNA Pol I
- Eukaryotic Pol E (epsilon)
- Both involved in PRIMER REMOVAL

Front 61

Why is DNA replication thermodynamically favorable? (4 conditions)...Drive polymerase reaction

Back 61

1. dNTPs --> dNMPs + PPi
2. PPi --> 2 Pi (loss of product)
3. Base stacking
4. Hydrogen bond formation (adds energy)

Front 62

What is the first step of the replication mechanism?

Back 62

1- Initiation at ori (origin of replication)

Front 63

What sequences originate the DNA strand?

Back 63

- Tandem array of three 13 bp sequences

Front 64

What is the significance of the 4 9bp sequences in the DNA

Back 64

Binding sites for DnaA protein

Front 65

What proteins promote unwinding at ori?

Back 65

dsDNA-binding proteins

Front 66

Function of ssDNA-binding proteins

Back 66

Keep separated ssDNAs in an extended form
- binds to DNA and prevents strands from meeting

Front 67

Function of helicase

Back 67

- Uses ATPs to mechanically invade and separate strands at replication fork

Front 68

Function of primase

Back 68

- Synthesizes short RNA primers of 3-20 nucleotides in length

Front 69

Direction of synthesis, free hydroxide

Back 69

Synthesis is in the 5' to 3' direction, giving free 3'-OH for subsequent nucleotide extension

Front 70

What is the name of primases in eukaryotes?

Back 70

Prokaryotes- primase
Eukaryotes- DNA Pol alpha is RNA-synthesizing primase

Front 71

Why is Pol alpha primase different from other DNA polymerases?

Back 71

- With exception of Pol alpha, DNA polymerases cannot initiate a new strand (de novo)
- Only elongate a pre-existing primer

Front 72

Function and kinds of DNA polymerases

Back 72

- Elongates primer with deoxynucleotides
- DNA Pol alpha, beta, gamma, delta, epsilon

Front 73

DNA Pol alpha function

Back 73

Primes both strands

Front 74

DNA Pol beta

Back 74

functions in repair fo nuclear DNA
- incorporates one nuc and leaves

Front 75

DNA Pol gamma

Back 75

carries out all mitochondrial DNA synthesis

Front 76

DNA Pol delta

Back 76

replication elongation of both strands (very processive)
- DOES MOST OF WORK!

Front 77

What is processivity?

Back 77

Avg number of nucleotides added before the enzyme dissociates from the DNA

Front 78

DNA Pol epsilon

Back 78

involved in repair of nuclear DNA and probably primer removal, using 5' to 3' exonuclease activity

Front 79

Prokaryotic DNA polymerases

Back 79

DNA Pol I
DNA Pol II
DNA Pol III

Front 80

DNA Pol I

Back 80

- repair synthesis and primer removal with 5' to 3' exonuclease activity
- First polymerase to be characterized

Front 81

DNA Pol II

Back 81

- participates in repair

Front 82

DNA Pol III

Back 82

- replication elongation (highly processive)
- Does most of replicative work in bacteria

Front 83

Topoisomerase I

Back 83

1- Cleaves one strand of parental DNA beyond forks
2- permits further unwinding of parental DNA
3- reseals (rejoins) the nicked ends

Front 84

Topoisomerase II

Back 84

- cleaves both strands
- allows unwinding of overwound Watson-Crick strands
- rejoins ends

Front 85

What is the cofactor for Topo II?

Back 85

- ATP

Front 86

What is a version of Topo II in bacteria?

Back 86

Gyrase
- makes a double strand scission
- forcibly underwinds DNA
- reseals scision

Front 87

What is result of undwerwining strain in gyrase?

Back 87

- Relieved by supercoiling

Front 88

Is topo I or II more important for bacteria?

Back 88

- Topo II is more crucial to the viability of the cell