Biochem Ii - Lecture 4 & 5

Front 1

T/F: The surprising intracy of DNA replication compared to the chemically similar transcription process arises from the need for extreme accuracy in DNA replication so as to preserve the integrity of the genome from generation to generation.

Back 1

True

Front 2

T/F: Nearly all known DNA polymerases can only add a nucleotide donated by a nucleoside triphosphate to the free 3'-OH group of a base paired polynucleotide so that DNA chains are extended only in the 5'->3' direction.

Back 2

True

Front 3

What is occasionally referred to as DNA's tertiary structure?

Back 3

Supercoiling

Front 4

What is the equation for supercoiling?

Back 4

L = T + W

Front 5

What is the linking number?

Back 5

The number of times that one DNA strand winds about the other; cannot change so long as the two strands are covalently attached in a circle

Front 6

What is the twist?

Back 6

Technically, the number of revolutions that one polynucleotide makes about the duplex axis; literally, the number of base pairs divided by 10.5

Front 7

What is the writhing number?

Back 7

The number of times the DNA helix crosses itself

Front 8

T/F: One single-strand nick by an endonuclease is enough to relax a supercoiled DNA.

Back 8

True

Front 9

T/F: Gel electrophoresis separates similar molecules on the basis of their compactness, so that the rate of migration of a circular duplex DNA increases with its degree of superhelicity; the agarose gel electrophoresis pattern of population of chemically identical DNA molecules with different linking numbers therefore consists of a series of discreet bands.

Back 9

True

Front 10

DNA in physiological solution has _ bp per turn.

Back 10

10.5

Front 11

The supercoiling of DNA is controlled by a remarkable group of enzymes known as...

Back 11

DNA topoisomerases (so named because they alter the topological state (linking #) of circular DNA but not its covalent structure.

Front 12

How do Type I and II topoisomerases differ from each other?

Back 12

Type I makes transient single-stranded breaks in DNA, while Type II makes double stranded breaks in DNA with the accompanying hydrolysis of ATP --> ADP + P(i)

Front 13

Type _ topoisomerases catalyze the relaxation of supercoils in DNA by changing their linking number in increments of one turn until the supercoil is entirely released.

Back 13

I

Front 14

Type IA topos only relax only...

Back 14

Negatively supercoiled DNA (it is a nicking-closing enzyme)

Front 15

When type IA topo is heated to denature it in the process of relaxing DNA, what intermediate is found?

Back 15

A topo-DNA (phosphotyrosine) intermediate

Front 16

What mechanism does type IA topo operate by?

Back 16

A strand passage mechanism

Front 17

What is the mechanism by which type IB topo's operate?

Back 17

A "nick and rotate mechanism"

Front 18

T/F: Type IB topoisomerases are said to mediate a controlled rotation mechanism in relaxing supercoiled DNA; this unwinding is driven by the superhelical tension in the DNA and hence requires no other energy input.

Back 18

True

Front 19

Why is DNA gyrase unique among topoisomerases?

Back 19

Because it generates negative supercoils in DNA

Front 20

T/F: Naturally occurring DNA's negatice supercoiling promotes DNA unwinding, i.e. it's favorable.

Back 20

True

Front 21

In what direction is new DNA ALWAYS replicated?

Back 21

5' --> 3'

Front 22

Which strand in DNA replication goes toward the direction of the replication fork, and which goes away?

Back 22

The leading strand goes toward the replication fork, and the lagging strand goes away from it in Okazaki fragments

Front 23

What are the two enzymes that can catalyze the formation of RNA primers called?

Back 23

RNA polymerase, and primase

Front 24

T/F: There are short RNA segments at the beginning (5' end) of each Okazaki fragment.

Back 24

True

Front 25

Name the general process that DNA Poly I follows in DNA replication.

Back 25

There is nucleophilic attack of the grown DNA chain's 3'-OH group onto the incoming nucleoside triphosphate, which eliminates PP(i) which is then hydrolyzed by pyrophosphatase to keep the reaction going.

Front 26

How does DNA Poly I "choose" the right base pairs to match with the template strand?

Back 26

DNA Poly I actually base pairs with the template strand, rather than directly recognizing the incoming base.

Front 27

T/F: Perhaps the simultaneous excision and polymerization activities of Pol I's 5' --> 3' activity protect DNA from the action of cellular nucleases that would further damage the otherwise gapped DNA.

Back 27

True

Front 28

What is the second and main function of the 5' --> 3' exonuclease function of DNA poly I?

Back 28

To remove the RNA primers from the Okazaki fragments

Front 29

What is nick translation?

Back 29

When there is a single-stranded nick in dsDNA, the 5' --> 3' exonuclease function of DNA polymerase I moves the nick towards the 3' end of the DNA, adding new base pairs along the way. (This is often utilized to yield radioactive DNA by placing only radioactive deoxynucleoside triphosphates in the solution to be incorporated into the nick translation)

Front 30

T/F: Pol I's 5'-->3' exonuclease also removes the RNA primers at the 5' ends of newly synthesized DNA while its DNA polymerase activity fills in the resulting gaps.

Back 30

True

Front 31

What enzyme acts as a participant in repairing DNA damage via the SOS reponse?

Back 31

DNA Polymerase II

Front 32

LECTURE: When determining the way that DNA is replicated by placing it first in dense N15 and then allowing it to replicate in lighter N14 and separating the DNA by density-gradient centrifugation, what results ultimately demonstrated that DNA is replicated semi-conservatively?

Back 32

After 1 generation, all strands were of an intermediate weight (1st gen had half heavy parent strand, half light daughter strand); after 2, some were of a very light weight (all new daughter light strands) and some were of the intermediate weight (still half parent and half daughter) and finally with more and more generations the DNA was at the light weight (more new daughter strands were forming with time).

Front 33

What would the density-gradient centrifugation have looked like if DNA replication were conservative? Dispersive?

Back 33

If conservative, the dense N15 parent strand would have never gone away; if dispersive, there would be a slew of bands of varying weights all across the plate

Front 34

T/F: DNA Polymerases have a "right hand" structure.

Back 34

True

Front 35

T/F: During the addition of a deoxynucleoside triphosphate to the 3' OH end of the daughter strand, one metal ion activates the primer's 3'-OH group for in-line nucleophilic attack on the incoming dNTP's alpha-phosphate group; and another metal ion acts to orient and electrically stabilize the negatively charged triphosphate group.

Back 35

Trueq

Front 36

Which function of which DNA polymerase excises mispaired nucleotides from the 3' end of the growing DNA srand?

Back 36

The 3' --> 5' exonuclease function of DNA poly I

Front 37

Which enzyme synthesizes a 3' primer for replication of DNA in Okazaki fragments?

Back 37

Primase

Front 38

What activity is used to remove RNA primers from the lagging strand Okazaki fragments?

Back 38

Nick translation

Front 39

Which enzyme ends nick translation by joining the 5' phosphate to the 3' OH of adjacent nucleotides on a DNA template?

Back 39

DNA ligase

Front 40

What are the 3 steps by which DNA Ligase uses NAD+ to catalyze the formation of a phosophodiester bond between the two open ends of a DNA nick?

Back 40

1. An adenyl group of NAD+ is covalently transferred to a Lys residue of ligase

2. The adenyl group is then transferred to the 5' phosphoryl terminus of the nick

3. The enzyme (ligase) catalyzes the formation of a phosphodiester bond on the open ends of the nick when the 3' OH group attacks the 5' phosphoryl group and closes the nick

Front 41

What is polymerase processivity dependent on?

Back 41

The presence of a B-subunit that is linked to the Poly III holoenzyme complex noncovalently but highly irreversibly

Front 42

What is the actual function of the Beta subunit?

Back 42

It serves as a molecular clamp to hold the polymerase onto the replication fork

Front 43

Since the beta subunit acts as a clamp and surrounds the entirety of the helix, it needs a machine to load it onto the helix. What does this?

Back 43

A clamp loader; or the lambda complex

Front 44

How is helicase activity different from topoisomerase activity?

Back 44

Helicase breaks the H-bonds between base pairs, and topoisomerase breaks the phosphate backbone

Front 45

What proteins in the Poly III holoenzyme prevent the DNA from rehybridizing after being broken apart by Helicase?

Back 45

SSB proteins

Front 46

When two forms of DNA are topologically equivalent, it means they are...

Back 46

Interconvertible without breaking any bonds

Front 47

Breaking or nicking the phosphate backbone of one DNA strand...

Back 47

Releases all of the supercoiling

Front 48

What does the gel pattern of negatively supercoiled DNA treated with Topoisomerase I ultimately indicate?

Back 48

That there is a favorable release in Energy, requiring no ATP, when Topo I releases the negative supercoils (think of the rubberband analogy)

Front 49

How does the Topo I enzyme act? Why?

Back 49

As a hinge and clamp, which allows the passage of the complementary strand through the single-stranded nick, increasing the Linking number until w = 0 and there is no more supercoiling

Front 50

Which Topo alters W by 2 and increases negative supercoiling?

Back 50

Topo II

Front 51

Describe the termination of the replication of a bacterial chromosome. What protein is required for the termination of replication?

Back 51

There are 6 termination sites; A, D, and E are right and opposite of oriC and only allow clockwise replication through it; B, C, and F are left and opposite oriC and only allow counter-clockwise replication. The Tus protein is required and binds to these sites and blocks the action of DnaB helicase.