Exam 2 Flashcards

Question

polytene chromosomes

Answer 1

* some dipterans (like Drosophila melanogaster, the fruit fly) have huge chromosomes in _interphase_ -found in the salivary gland cells * generated by **successive replications** of a chromosome _without separation_ of the replicated chromosomes in mitosis

Answer 2

* can label gene-specific probes to identify where specific genes are on the banding

Answer 3

* sites of gene expression/activity on polytene chromosomes expand to give "puffs" * show that gene expression requires that the DNA must _unwind_

Answer 4

pairs of chromosomes similar in size, shape and gene content

Answer 5

one of the two DAN strands in a _replicated_ chromosome

Answer 6

chromatids from the same chromosomes - join at the centromere

Answer 7

chromosomes replicate

Answer 8

chromosomes/sister chromatids condense and attach at the kinetochore to the mitotic spindle microtubules

Answer 9

sister chromatids line up in center

Answer 10

sister chromatids pulled to opposite opposite poles of the cell

Answer 11

sister chromatids in opposite poles and nuclear membrane reforms

Answer 12

membrane pinches in middle to separate into two daughter cells - cytoplasm is divided

Answer 13

proteins that hold "glue" together sister chromatids

Answer 14

* a constricted region of a chromosome (the DNA) that: - has unique DNA sequences and proteins not found anywhere else in the chromosome * is where the sister chromatids attach to the mitotic spindle microtubules * centromere region also contains the kinetochore

Answer 15

the proteins responsible for attaching to the spindle apparatus microtubules

Answer 16

DNA is wrapped around: - normal histone H3 - or a centromere-specific histone H3 variant, Cen-H3 * Cen-H3 allows binding of kinetochore proteins to form the kinetochore * also has heterochromatin that is rich in satellite DNA sequences (repetitive DNA) -function of the repetitive centromeric DNA is not known

Answer 17

1. supercoiled DNA must first be relaxed 2. initiation 3. elongation 4. joining and/or termination

Answer 18

higher organisms with _linear chromosomes_ * have long series of short tandem repeated sequences called telomers -may be 100-1000 repeats * human telomere repeats are -TTAGGG-3'

Answer 19

* DNA replication leaves a 3' unreplicated end on one of the replicated DNA strands

Answer 20

* DNA replication leaves one strand with unreplicated end * next round of replication results in a shorter DNA -due to shorter DNA template * and so on until genes near the ends are not replicated

Answer 21

**telomerase** uses: * the _free 3'-OH_ of the telomeric strand * its own _RNA template_ of 3'-AAUCCC-5' * a _reverse transcriptase_ * adds tandem repeats (5'-TTAGGG-3' in humans) to the 3' end at each chromosomal terminus * extends the ends of the chromosomes to solve the so-called end replication problem * telomerase uses RNA to complementary bind to end of telomer * then the RNA polymerase of telomerase adds the complementary dNTPs to extend the end

Answer 22

* repeat this step many times until it is long enough for the DNA polymerase to do another Okazaki fragment * then DNA polymerase can fill in the gap RESULT * extends the end of the chromosome back to how long it should be

Answer 23

telomerase is expressed: * in actively dividing cells -stem cells -during development * not expressed in quiescent cells -most other cells in the body * loss of telomeres results in senescence -cell dies * cancer cells often have telomerase

Answer 24

* can be recognized by the DNA repair enzymes as a broken chromosome * could be added to the end of another chromosome * must have a way to prevent this!

Answer 25

the protein **TRF2** * allows the 3' telomer unit to _invade into its homolog_ in an upstream region of the telomere * forms the **t-loop** * t-loop prevents _DNA repair enzymes_ from recognizing the 3' end as a DNA break

Answer 26

**(TRF1, TRF2, Rap1, TIN2, TPP1, and POT1)** * form the **Shelterin** complex 1. function to protect the telomers from DNA damage repair * can result in chromosome ends sticking to other chromosomes 2. also function to control telomer length by inhibiting telomerase * the more shelterins bind, the less telomerase can bind to add more DNA

Answer 27

* a unit of the genome in which DNA is replicated * bacteria usually only have one replicon * eukaryotes can have many replicons * each replicon contain an _origin for initiation of replication_ **origin**- a sequence of DNA at which replication is initiated **terminus** - a segment of DNA at which replication ends

Answer 28

* grow organism on "heavy" ¹⁵N to label DNA * then grow on medium with "light" ¹⁴N * allow DNA replication to occur * isolate DNA and ultracentrifuge in dense medium

Answer 29

**parental DNA** - all heavy DNA **1st generation** - all medium hybrid DNA **2nd generation** - mixture of light and medium DNA _suggests DNA is replicated semiconservatively_ - resulting replicated DNA strand is one parental and one new

Answer 30

* replication accomplished by **separation of the strands of a parental duplex** * with each strand then acting as a template for synthesis of a complementary strand

Answer 31

in electron microscopy: * a replicated region appears as a bubble within nonreplicated DNA * a replication fork is initiated at the origin and then moves sequentially along DNA

Answer 32

when a single replication fork is created at an origin

Answer 33

when an origin creates _two replication forks_ that move in opposite directions

Answer 34

* bacterial replicons are usually **circles** * they replicate **bidirectionally** from a **single origin** * the origin of *E.coli* is **oriC** -245 bp in length

Answer 35

cells cycle between: * **mitotic (M) phase** = when cells actually divide * **interphase** = the non-dividing phase -the chromosomes are generally uncoiled in euchromatin and heterochromatin **cells spend most of their time in interphase**

Answer 36

**only during interphase** - not during mitosis **DNA synthesis occurs in the S phase** of interphase

Answer 37

**interphase** has **two gap phases** - no DNA synthesis occurs - G1 and G2

Answer 38

**cell growth** * everything in the cell is doubled except DNA * cells prepare for DNA synthesis * make all enzymes for DNA synthesis

Answer 39

- after DNA synthesis - preparing for mitosis **organelles replicate and additional cytoplasm is made in G1 and G2**

Answer 40

progression from G1 into S phase is tightly controlled **checkpoint** a control mechanism that prevents the cell from progressing to the next stage unless specific goals and requirements have been met * no damage to the DNA * must be a certain amount of cell growth

Answer 41

* too much DNA for just one origin/replicon * not just DNA, but DNA + histones * eukaryotic replicons are 40 to 100 kb in length * _multiple origins of replications_ that ultimately merge during replication

Answer 42

**S phase** * early replicating * late replicating * see ~100 to ~300 replication foci when stained * eukaryotic replication takes over 6 hours to complete -S phase

Answer 43

~400 kDa protein complex * highly evolutionarily conserved in eukaryotes * binds to eukaryotic origin * is **cis-acting** causes DNA replication initiation on the strand of DNA to which it binds

Answer 44

* inject nucleus into a *Xenopus* (frog) egg * DNA replicates * but it cannot replicate again -occurs only once * but if you permeabilize the nuclear membrane before injection -DNA will be replicated more than once

Answer 45

allows _new protein_ made in the cytoplasm to enter the nucleus and initiate replication again if you permeabilize before injection: * suggests that a _protein_ is found in the nucleus that allows initiation of replication * once replication occurs, the protein is used up or inactivated

Answer 46

controls eukaryotic re-replication * is necessary for initiation of replication at each origin * present in the nucleus **prior to replication**, but is removed, inactivated, or destroyed by replication

Answer 47

* active licensing factor is present in the nucleus * replication inactivates it * new licensing factor is made in the cytoplasm * but it cannot get into the nucleus through the nuclear membrane * breakdown of the nuclear membrane in mitosis allows new licensing factor to associate with the nuclear material

Answer 48

* **cyclins and cyclin-dependent kinases (Cdk)** * they are _Helicase Activating Proteins_ * Cdk enzymes _must bind cyclins_ to be active -becomes the "ON-OFF" switch

Answer 49

the enzyme that separates the strands of DNA for replication - a crucial initial step for replication

Answer 50

* relieves stress coiling * relaxes the supercoil * moves in front of the replication fork

Answer 51

ssDNA nick to allow cut strand to rotate around the uncut strans

Answer 52

both strands of DNA are cut and the intact strand is passed through the cut to relieve the tension

Answer 53

* origin recognition complex binds to the origin * DNA strands must be separated and stabilized -creates replication bubble * DNA synthesis is initiated at the replication forks

Answer 54

**replisome** multiprotein complex that assembles at the replication fork to undertake synthesis of DNA * it contains DNA polymerase and other enzymes * is assembled *de novo* (at that time and place) into the replisome complex * replisome moves along the DNA as the DNA is unwound * daughter strands of DNA are synthesized

Answer 55

* joining of replication forks or termination of DNA synthesis * separation of the duplicated chromosomes

Answer 56

1. DNA-dependent DNA polymerase (or DNA polymerase) 2. DNA Repair Polymerase

Answer 57

* also known as **DNA replicase** * is only one subunit of a large protein assembly called the **holoenzyme** * responsible for **semiconservative replication**

Answer 58

* responsible for _excising damaged DNA bases_ * then _synthesizing new DNA to replace_ the excised DNA

Answer 59

* synthesize DNA antiparallel from 5' to 3' * require a _template_ that is 3' to 5' -nucleotide choice is determined by complementary base pairing with the template * require a free 3'-OH to add nucleotides to * polymerase makes the phosphodiester bond

Answer 60

* bacterial (& eukaryotic) cells have several different **DNA polymerase** enzymes * **DNA polymerase III** - is the bacterial DNA polymerase * does semiconservative replication

Answer 61

**DNA polymerase I** * repair of damaged DNA **DNA polymerase II** * required to restart a replication fork when the process is stopped by DNA damage **DNA polymerase IV and V** * allow replication to bypass some types of DNA damage * error-prone polymerases

Answer 62

* only polymerase that can do **Nick Translation** * DNA polymerase I has a _5'-3' exonuclease activity_ * can remove nucleotides _in front of the polymerase_ * it can also recognize a _single strand nick_ in the DNA * inserts a 5'-3' exonuclease removes nucleotides ahead of the polymerase as it makes new DNA

Answer 63

* DNA polymerase I cleaved with an enzyme _yields 2 parts:_ 1. larger fragment = polymerase + proofreading enzyme * used in the laboratory to synthesize DNA 2. smaller fragment is the 5'-3' exonuclease

Answer 64

* how well does the polymerase copy the DNA? * substitutions = inserting the wrong base * frameshifts = inserting an extra nucleotide or deleting a nucleotide * DNA polymerases have a **proofreading ability** -due to a **3' to 5' exonuclease** * is used to excise incorrectly paired bases -detects A-C or A-A mispairing

Answer 65

* dNTP binds to template base by complementary binding * polymerase catalyzes joining of phosphate to 3'OH of primer * using energy of pyrophosphate release

Answer 66

* if wrong base is inserted and added to the chain -causes a _warp in the chain_ that _slows_ the polymerase * allows the 3'-5' exonuclease to back up and remove that wrong base * polymerase then has the chance to add the right base * the fidelity of replication is improved from proofreading by a **factor of ~100**

Answer 67

* polymerase to synthesize DNA 5'-3' * 3' to 5' exonuclease for proofreading * 5'-3' exonuclease that can allow Nick Translation -removes bases in front of the polymerase (*note*: all bacterial DNA polymerases have the 3'-5' proofreading exonuclease)

Answer 68

**the right hand** * DNA lies across the "palm" -catalytic site * a groove is created by the "fingers" and "thumb" * DNA slides through the groove as synthesis continues

Answer 69

**leading strand synthesis** * DNA synthesis goes 5'-3' * DNA pol advances **continuously** when it synthesizes the leading strand

Answer 70

* DNA synthesis MUST go 5'-3' * as DNA synthesis on the lagging strand beings -the leading strand is still moving ahead -this leaves a gap on the lagging strand -so the pol must bind again near the fork and start a new strand

Answer 71

* lagging strand is _synthesized in many fragments_ * called **Okazaki fragments** * these Okazaki fragments must then be connected together

Answer 72

separates (or melts) the DNA duplex - E.coli has 12 different helicases - most are multimeric - most common is a Hexamer

Answer 73

* helicase encircles a ssDNA strand * alternates between a dsDNA binding conformation and a ssDNA binding conformation * this separates the DNA strands * requires the energy of ATP hydrolysis

Answer 74

must: * protect the ssDNA * prevent it from re-annealing with the other strand * **leading strand** DNA is synthesized quickly -no real need to protect the ssDNA * **lagging strand DNA** _must wait_ until there is sufficient space to begin an Okazaki fragment synthesis -must protect this ssDNA

Answer 75

* SSBs bind to the lagging strand ssDNA after unwinding by helicase * SSB binding is **cooperative** -binding of one SSB enhances the binding of subsequent SSBs

Answer 76

* all DNA pol require a 3'-OH priming end to initiate DNA synthesis

Answer 77

* small RNA polymerase -makes RNA 5'-3' -does not require a free 3'-OH * makes an RNA primer for DNA synthesis * associates with the oriC complex in bacteria -which binds to the origin * recognizes the origin and synthesizes ~10 bases of RNA complementary to the DNA at the origin

Answer 78

requires: * binding of the helicase to unwind the DNA * binding of SSBs to protect and prevent re-annealing * primase binds and makes a complementary 10 bases of RNA

Answer 79

* leading strand is synthesized _continuously_ * lagging strand is synthesized _discontinuously_ - polymerase must continually detach and move back to the replication fork * *E.coli* uses the same polymerase for both (circular chromosome) - eukaryotes use different enzymes for leading and lagging strands

Answer 80

* **2-DNA polymerase III catalytic cores** -a _catalytic subunit_ (alpha) -and a 5'-3' _proofreading subunit_ * **2 clamps** -ensures processivity = that the polymerase stays with the DNA * **a clamp-loader/dimerization** complex -loads the DNA into the clamps -brings the 2 polymerase complexes together

Answer 81

* a **clamp loader** complex assists in loading the circular **clamp** around the DNA strand * catalytic **core polymerase** associates with each template strand * the **dimerization** subunits of the clamp loader helps in dimerization of the **2** core polymerases * the **clamp loader** stays associated with the polymerase on the lagging strand * it will be needed to reload the polymerase after each Okazaki fragment

Answer 82

* the clamp forms a ring around the DNA * holds the polymerase strongly to the DNA - makes the polymerase highly **processive** = stays with the DNA * the clamp associated with the polymerase on the lagging strand dissociates at the end of each Okazaki fragment and reassembles for the next fragment

Answer 83

see ch. 11 notes

Answer 84

* leading strand continuing to replicate DNA * lagging strand -newly synthesized Okazaki fragment terminate just before the previous Okazaki fragment -must remove the RNA primer -synthesize DNA to replace the RNA primer -seal the last phosphodiester bond between the Okazaki fragment

Answer 85

- DNA polymerase III dissociates leaving a gap and the RNA primer - **DNA pol I** uses the "nick" to attach and uses its 5'-3' exonuclease to remove the RNA primer and replace with DNA

Answer 86

* DNA pol I is released leaving a "nick" between the Okazaki fragments * **DNA ligase** seals the remaining _phosphodiester bond_ at the nick * DNA ligase uses energy from ATP to catalyze the reaction

Answer 87

eukaryotes have **3 polymerases** for DNA synthesis * **DNA polymerase alpha/primase** initiates synthesis of new strands -has _DNA pol_ activity -and _RNA pol_ activity for _making its own primer_ * DNA polymerase ε synthesizes the _leading strand_ * DNA polymerase δ synthesizes the _lagging strand_ * all 3 are linked in the replisome

Answer 88

* **helicase (MCM)** binds the DNA * **pol α/primase** detaches (but remains with replisome) * **clamo loader (RFC)** mediates binding of the clamp (PCNA) to the ssDNAs * clamp and pol δ release to make another Okazaki fragment * but in subsequent okazaki fragments, pol δ continues and into the RNA primer

Answer 89

- polδ displaces the RNA primer - leaving a flap of RNA - **FEN1** nuclease removes the RNA primer flap - polδ then _fills the gap_ * **DNA ligase I** seals the remaining nick

Answer 90

DNA synthesis stops and replication fork may collapse _3 options_: * cell death (not really acceptable) * skip over the lesion = **lesion bypass** * repair by **recombination**

Answer 91

- the replication fork stalls - the replication complex must be replaced by _error-prone polymerases_ -DNA pol IV or V (for bacteria) -can copy through the error and incorporating the error - error-prone polymerases removed - restart DNA synthesis with **primosome** -primase and polymerase complex that restarts synthesis * then primosome is replaced by the replisome

Answer 92

- excise damage - information from the undamaged other strand is used to repair the damaged sequence - DNA synthesis is restarted - DNA repair is used to fix the gap later

Answer 93

bacteria: - the two replication forks meet halfway around the circle - *ter* sites that cause termination if the replication forks go too far

Answer 94

eukaryotes: - telomer region - leading strand completes replication - lagging strand has unreplicated end

Answer 95

includes: 1. direct reversal of DNA damage 2. base excision repair - remove the base and replace with appropriate base 3. nucleotide excision repair - remove and replace the whole nucleotide 4. mismatch repair - requires determination of "new" and "old" DNA strands

Answer 96

- repair mechanism that retrieve an undamaged sequence from the other replicated DNA strand - nonhomologous end joining - translesion/error-prone repair

Answer 97

- missed by the 3'-5' proofreading exonuclease - results in a _major distortion_ of the DNA strands - repaired by the **mismatch repair system** -if not, becomes a permanent change in the DNA

Answer 98

e.g. spontaneous deamination of _cytosine to uracil_ -can also happen to other bases * creates a mismatched U-G pair * results in a _minor structural distortion_ of the DNA * uracil is preferentially removed by **base excision repair**

Answer 99

**depurination** * _spontaneous_ loss of a base by hydrolysis * blocks _DNA replication and transcription_ * corrected by **nucleotide excision repair system**

Answer 100

- substances that increase the rate of mutation by inducing changes in the DNA sequence - either directly or indirectly

Answer 101

a substance that promotes the formation of cancer

Answer 102

- UV light at ~260nm is absorbed by the bases - can cause a **photochemical fusion** of two _pyrimidine_ bases side-by-side - **thymine-thymine dimer** -covalent bonds - blocks replication and transcription - corrected by **nucleotide excision repair**

Answer 103

- includes mutagens like **nitrosamines** - found in tobacco products or produced from some food preservatives like nitrates e.g. methylation of guanine - creates a bulky base that distorts the DNA - inhibits replication and transcription

Answer 104

used for direct reversal - transfers the methyl group to the methyltransferase

Answer 105

* oxidizing agents generated by ionizing radiation or chemical agents * generates **free radicals** - superoxides, hydroxyl radicals, hydrogen peroxide, etc. * oxidation of guanine * causes base pairing with adenine * correct by **mismatch repair**

Answer 106

- gamma radiation or X-rays - directly ionizes the deoxyribose (indirectly causes reactive oxygen species) - causes **double strand DNA breaks** - kills rapidly proliferating cells (cancer treatment) - corrected by **recombinant repair**

Answer 107

- alkylating agents resulting in methylation of guanine to methyl-guanine - _methyltransferase_ - transfers the methyl group to the methyltransferase

Answer 108

* for _spontaneous base deamination_ - cytosine deamination to uracil * also _alkylated bases_ * causes the removal of an _individual damaged base_ * **glycosylases** cleave the _bond between the deoxyribose and the base_ * results in a removal of a base

Answer 109

* eukaryotic DNA pol specific for base excision repair * replaces the nucleotide

Answer 110

- removes mispaired or damaged bases then synthesizes a new stretch of DNA to replace them - used for almost all excision repair in *E.coli*

Answer 111

* **UvrAB** dimer recognizes the damage * **UvrA** is released * **UvrC** joins UvrB * **UvrBC** cuts DNA strand above and below the damage * **UvrD** (helicase) then unwinds DNA * DNA pol I then binds * removes the damaged area as it synthesizes new DNA * DNA ligase seals the nick

Answer 112

**Xeroderma pigmentosum (XP)** - a human disease caused by mutations in any one of several nucleotide excision repair genes - hypersensitivity to sunlight and UV light - results in skin diseases and cancers - disease was helpful in understanding the excision repair system

Answer 113

1. global genome repair 2. transcription-coupled repair

Answer 114

XPC protein detects the damage and initiates repair

Answer 115

- damage is recognized by the RNA polymerase during transcription - RNA pol stalls at the damage - the RNA pol is released

Answer 116

* **TF_IIH** transcription factor complex binds * _helicase_ in complex unwinds the DNA * complex _endonucleases_ cut DNA above and below damage * DNA pol δ/ε -eukaryotic replisome polymerases * excises and replaces the gap * DNA ligase seals the nick

Answer 117

- purpose is to repair mismatches immediately after DNA replication - relies on **MutS** complex - _dimer_ that embraces the DNA - moves along the DNS looking for _distorted DNA_ due to mismatched bases

Answer 118

- after DNA synthesis in bacteria - **Dam Methylase** methylates DNA at -**GATC**- sequences all along the DNA - but shortly after synthesis, new DNA is **not** methylated - so unmethylated DNA would have the mistake -the original template DNA would be methylated

Answer 119

see ch. 14 notes

Answer 120

- similar to *E. coli* - eukaryotic DNA is methylated BUT mismatch repair DOES NOT use methylation to identify the new strand

Answer 121

- mismatch repair is _coupled_ to the DNA synthesis machinery - uses nicked Okazaki fragments to ID new strands - also associated with clamp to determine the new strand - then uses a similar mechanism to remove and replace the mismatch nucleotide

Answer 122

patterns of inheritance - single genetic locus or just a few loci

Answer 123

an organisms complete set of DNA, including all of its genes - also includes mitochondrial and chloroplast genomes - extrachromosomal DNA and other forms of inheritance

Answer 124

genome-wide studies via DNA or RNA sequencing + bioinformatics

Answer 125

main principle: _chain termination_ method based on terminating DNA synthesis one nucleotide at a time components: 1. DNA primers (only one per sequencing rxn) 2. DNA pol 3. nucleotides (dNTPs) 4. labeled modified nucleotides dideoxynucleotides (ddNTPs)

Answer 126

chain-terminating nucleotides, lacking a 3'-OH group required for the formation of a phosphodiester bond during DNA elongation. incorporation of a dideoxynucleotide into the elongating DNA strand therefore terminates the extension, resulting in DNA fragments of varying length

Answer 127

- an easier way - sanger method based - non-radioactive - more automated

Exam 2 Flashcards

(154 cards)