Textbook 1.2

Question 1

DNA Gyrase

Answer 1

Type of topoisomerase that relieves mechanical strain that builds up during replication in E. coli (super coiling). Cleaves DNA duplex in two and passes one of the strands through. Eukaryotes have similar enzymes which have same function.

Question 2

For DNA polymerase to work…. must be present

Answer 2

DNA and all four deoxyribonucleoside triphosphates dTTP, dATP, dGTP, dCTP

Question 3

All DNA polymerases CANNOT…and thus require …

Answer 3

• initiate the formation of a new DNA strand—can only add nucleotides onto the 3’ OH terminus of an existing strand
• a template strand to copy and a primer to provide the OH end

Question 4

What is the major enzyme responsible for DNA replication…

Answer 4

DNA Polymerase III, even though there is far fewer copies of the enzyme than Polymerase I which was discovered first.

Question 5

Replication is said to be…

Answer 5

semi-discontinuous

Question 6

Which enzyme links Okazaki fragments together

Answer 6

DNA ligase

Question 7

Which enzyme initiates DNA replication

Answer 7

An RNA polymerase called RNA primase which constructs a short primer of RNA

Question 8

What happens to the RNA primers at the end of replication?

Answer 8

They are removed and replaced with DNA. The strands are then sealed by DNA ligase.

Question 9

What is a possible reason for an RNA primer being used?

Answer 9

Potentially more mistakes made during initiation than elongation so a short, removable RNA primer avoids inclusion of mismatched bases.

Question 10

Helicase

Answer 10

Unwinding enzyme that uses energy from hydrolysis of ATP to move along strand. Forms a ring like structure around strand.

Question 11

SSB Proteins

Answer 11

Single-stranded DNA-binding proteins: bind selectively to single stranded DNA to keep it extended and prevent it from rewinding or getting damaged.

Question 12

Primosome

Answer 12

Transient association between primase and helicase in bacteria during replication.

Question 13

Describe the polymerase complex during replication

Answer 13

Both the leading and lagging strand have separate DNA polymerases which are adding onto the 3’ OH terminus of the leading and lagging strand. The DNA polymerase on the leading strand is synthesized continuously while the protein on the lagging strand synthesizes in chunks then releases template to move along to the next fragment. It is hitching a ride on the other polymerase. They are part of one complex even though there are two polymerases because the DNA of the lagging strand template is looped back on itself. They are moving towards opposite ends of their respective templates but ultimately still in same direction.

Question 14

DNA polymerase III holoenzyme

Answer 14

replication machine. Includes:

• DNA polymerase III
• beta clamp (keeps polymerase associated w template)
• clamp loader (holds clamp open for DNA to squeeze into ring of beta clamp)

Question 15

Difference between beta clamp and DNA polymerase III association on the leading and lagging strand

Answer 15

Polymerase on the leading strand remains tethered to the beta clamp for the entire duration of the replication while polymerase on the lagging strand disengages from the beta clamp after completing an Okazaki fragment and is cycled to a new beta clamp on an RNA primer-DNA template junction closer to replication fork.

Question 16

Exonucleases can be divided into…

Answer 16

5’ to 3’ and 3’ to 5’ depending on the direction of the degradation of the DNA strand

Question 17

Why is DNA polymerase I almost like three enzymes in one?

Answer 17

It has polymerizing capabilities but also 3’ -> 5’ and 5’ -> 3’ exonuclease capabilities. These activities are found in three different domains of the polypeptides. The 5 to 3 exonuclease is not RNA/DNA specific unlike most other exonucleases and can degrade both.

Question 18

Which enzyme removes the RNA primers that initiate okazaki fragments?

Answer 18

5’ to 3 ‘ exonuclease activity of DNA polymerase I. It also fills the gaps as it removes the primers.

Question 19

Which enzyme seals okazaki fragments together

Answer 19

DNA ligase

Question 20

How does DNA polymerase ensure the right nucleotides are being added?

Answer 20

Bond angles and geometry of base pairs. If the nucleotide has the proper fit the enzyme will undergo a conformational change to add onto the growing chain. If it does not have proper geometry enzyme cannot achieve conformation required for catalysis.

Question 21

What happens if the wrong nucleotide is added?

Answer 21

The frayed end of the 3’ strand is fed into the 3’ to 5’ active nuclease site (at wrist) where the incorrect base is removed. This removes 99 out of every 100 mismatched bases.

Question 22

Fidelity of DNA replication relies on…

Answer 22

1. accurate selection of nucleotides
2. immediate proofreading
3. post-replicative mismatch repair

Question 23

Human genome contains about

Answer 23

21 000 protein coding genes, same number as microscopic worm whose entire body has only about 1000 cells.

Question 24

90% of human genes participate in…

Answer 24

alternative splicing which allows for one gene to encode a number of related proteins.

Question 25

What can explain the difference in complexity between organisms?

Answer 25

It’s less a result of amount of genetic information in an organism’s genome and more the manner with which it is put to use.

Question 26

FOXP2

Answer 26

Difference in two amino acids between chimp and human version of this protein. Mutations of this gene cause severe speech impairment in humans. Possible speech gene?

Question 27

SRGAP2

Answer 27

duplicated in humans, increases number of dendritic spines (may have contributed to our brain size) .

Question 28

AMY1

Answer 28

encodes for starch digesting enzyme amylase. Humans have more copies on average (amount is variable).

Question 29

Highly repeated DNA sequences

Answer 29

constitutes 1-10% of total DNA. Given sequence repeats itself over and over with no interruptions. Includes satellite DNA, mini-satellite DNA, and micro-satellite DNA.

Question 30

What is mini-satellite DNA used for?

Answer 30

DNA fingerprinting and identifying individuals in criminal/ paternity cases. This DNA is highly variable from individual to individual.

Question 31

Where is satellite DNA found?

Answer 31

It’s localized in the centromeric regions of chromosome

Question 32

FISH

Answer 32

Fluorescence in situ hybridization

- utilizes satellite DNA and re-annealing capabilities to label DNA fragments

Question 33

Moderately repeated DNA sequences

Answer 33

Can constitute 20-80% of total DNA depending on organism. Interspersed throughout genome. Does not occur in tandem sequences (repeats right after another).. Includes SINEs and LINEs.

Question 34

Non repeated DNA sequences

Answer 34

localize to a particular region on a particular chromosome and includes DNA sequences that code for virtually all proteins other than histones.

Question 35

DNA of prokaryotes is not…

Answer 35

packaged into chromatin, so DNA binding proteins can bind directly to preferred binding sites

Question 36

Nuclear envelope

Answer 36

Biggest distinction between prokaryotes and eukaryotes. Forms boundary between nucleus and cytoplasm and consists of two cellular membranes which are fused at sites to form circular pores.

Question 37

In the typical interphase (non-mitotic) cell, the nucleus contains…

Answer 37

chromosomes as chromatin, nucleoli (electron dense structures), and nucleoplasm

Question 38

Nuclear Lamina

Answer 38

provides mechanical support to nuclear envelope, site of attachment for chromatin fibres at the nuclear periphery. Filamentous and meshy.

Question 39

Heterochromatin does not extend…

Answer 39

into the region of the nuclear pore

Question 40

Average human cell has…

Answer 40

6.4 billion bp divided among 46 chromosomes

Question 41

The larger the chromosome

Answer 41

the longer the strand of DNA that makes it up

Question 42

Chromatin

Answer 42

Stuff that makes up chromosomes—composed of DNA and associated proteins

Question 43

Histones contain an unusual amount of

Answer 43

arginine and lysine

Question 44

Histones

Answer 44

Responsible for orderly packing of eukaryotic DNA. There are five types: H1, H2A, H2B, H3, H4

Question 45

Which of the histone proteins have undergone the least evolutionary change?

Answer 45

H3 and H4, though none of them have changed that much because the positive charge of this peptide is responsible for associating with the negative backbone of the DNA molecule (same in all organisms).

Question 46

Nucelosomes

Answer 46

Contains a core particle consisting of 14 bp of supercoiled DNA wrapped almost twice around a histone octamer.

Question 47

What’s important about the N-terminal segments of histones?

Answer 47

They are targets of key enzymes that play a key role in making the chromatin accessible to proteins.

Question 48

Lowest level of chromatin structure

Answer 48

DNA molecule wrapped around nucleosome core particle of 10 nm diameter

Question 49

What have linker histones and core histones been implicated with?

Answer 49

Higher-order packing of chromatin. Without H1, 30 nm fibres uncoil to form beaded filament. Chromatin fibres without H4 histone tails cannot fold into higher order fibres.

Question 50

Cohesion

Answer 50

protein that maintains DNA loops when 30 nm fibre is coiled/looped up into thicker fibres

Question 51

Heterochromatin

Answer 51

chromatin that remains compact during interphase (little transcriptional activity)

Question 52

Euchromatin

Answer 52

dispersed, active state

Question 53

Constitutive heterochromatin

Answer 53

Remains in compacted state in all cells at all times (silenced DNA), usually found in regions that flank the telomeres and at centromere. When active genes move to this region they tend to become transcriptionally silent. Inhibits genetic recombination between homologous repetitive sequences.

Question 54

Facultative heterochromatin

Answer 54

Chromatin that has been specifically inactivated during certain stages of an organism’s life. Ex. only one of a female’s X chromosomes is transcriptionally active while the other is a heterochromatic clump (barr body).

Question 55

X-chromosome inactivation

Answer 55

1. Heterochromatinization of X chromosome occurs early in embryonic development
2. Paternally and maternally derived X chromosome have an equal chance of being inactivated in any given cell (any descendent cell after has the same X chromosome inactivated)
3. Reactivation of the Barr body occurs in female germ cells prior to onset of meiosis - both x chromosomes are active during oogenesis.

Question 56

Adult females are

Answer 56

genetic mosaics; different alleles function in different cells

Question 57

Where are inactive or less gene rich chromosomes found

Answer 57

periphery of nucleus