Biochem 3-5 Replication

Front 1

replication

Back 1

DNA synthesis

Front 2

transcription

Back 2

RNA synthesis

Front 3

translation

Back 3

protein synthesis

Front 4

In prokaryotes (organisms without nuclei such as bacteria) replication occurs in the _____.

Back 4

cytoplasm

Front 5

In eukaryotes (organisms with nuclei such as humans) replication occurs in the _____ during S phase of the cell cycle.

Back 5

nucleus

Front 6

The purpose for DNA replication is to duplicate _____, so that after mitosis each daughter cell will inherit a complete genome.

Back 6

chromosomes

Front 7

DNA replication only occurs in dividing cells. Non-dividing cells blocked in ___ do not progress to ___ phase, so they do not replicate their DNA.

Back 7

Go, S

Front 8

Characteristics of DNA Replication: Requirements

Back 8

DNA Polymerase, Mg+2, Template, Primer, dNTPs

Front 9

Characteristics of DNA Replication: Direction

Back 9

5'-3'

Front 10

Characteristics of DNA Replication: Complimentary

Back 10

For each A of the template strand, a T is added to the new strand, and each G is matched with a C.

Front 11

Characteristics of DNA Replication: Proofreading

Back 11

3' to 5' exonuclease activity removes mismatched nucleotides.

Front 12

Characteristics of DNA Replication: Bidirectional

Back 12

Replication proceeds in both directions from central origins of replication (ori).

Front 13

Characteristics of DNA Replication: Discontinuous

Back 13

The lagging strand is synthesized as short stretches.

Front 14

Characteristics of DNA Replication: Semiconserative

Back 14

One strand of a daughter chromosome is newly synthesized, the other template strand is inherited intact from the parental chromosome.

Front 15

DNA Polymerase (Pol#)

Back 15

An enzyme that catalyzes polymerization of dNTPs into DNA.

Front 16

What are the prokaryotic DNA polymerases?

Back 16

Pol I, II & III (I, II & III are also RNA Pols)

Front 17

What are the eukaryotic DNA polymerases?

Back 17

Pol δ, α, β & γ

Front 18

DNA Pol δ

Back 18

-Location: nucleus
-Fnx: leading strand synth
-Processivity: >100,000 bp
-Proofreading: +
-RNA primer: +

Front 19

DNA Pol α

Back 19

-Location: nucleus
-Fnx: lagging strand synth
-Processivity: ~180 bp
-Proofreading: -
-RNA primer: +

Front 20

DNA Pol β

Back 20

-Location: nucleus
-Fnx: repair gap filling
-Processivity: ~20 bp
-Proofreading: -
-RNA primer: -

Front 21

DNA Pol γ

Back 21

-Location: mitochondria
-Fnx: synth of both strands
-Processivity: >8300 bp
-Proofreading: +
-RNA primer: -

Front 22

Although the ___ ion is not incorporated into DNA, it is a cofactor required for DNA polymerase activity.

Back 22

Mg2+

Front 23

To synthesize a new strand, DNA polymerase must read a preexisting strand. This preexisting strand is known as the _____.

Back 23

Template

Front 24

Deoxyribonucleotides (dNTPs)

Back 24

building blocks of DNA. All four are required for replication.

Front 25

Remember that dNTPs lack _____ (hence the prefix deoxy). Thymine, which has a methyl group, is used instead of methyl free _____, which is used for RNA.

Back 25

2' hydroxyls, uridine

Front 26

Hydrolysis of the _____ provides the energy required for nucleotide polymerization.

Back 26

triphosphate bonds; Energy is stored in the triphosphate bonds as electrostatic repulsion of their negatively charged oxygens.

Front 27

The final requirement for DNA replication is a primer. This a free hydroxyl (OH) at the ___ position of the terminal nucleotide of a previously existing strand. A strand with a free ___ OH is also referred to as a primer.

Back 27

3', 3'; The reason for this requirement is DNA polymerase can only bind a 3' OH with the 5’ phosphate of an incoming nucleotide. The opposite reaction will not occur. The hydroxyl of a new nucleotide cannot be bound with the phosphate of a preexisting strand

Front 28

DNA is synthesized in the 5'-3' direction, therefore the template strand must be read _____.

Back 28

3'-5'

Front 29

Pol __ and Pol ___ have the abilit.y to “double check” whether each new nucleotide is complimentary to the corresponding base of the template.

Back 29

δ, γ; If an incorrect nucleotide is incorporated, the polymerase’s 3'-5' exonuclease activity “kicks back,” excising the mismatch. Pol α and Pol β lack this activity.

Front 30

How is possible for the lagging strand to be synthesized in the 3'-5' direction if no DNA polymerase is capable of doing so?

Back 30

Okazaki fragments; The dilemma is resolved by discontinuously synthesizing the lagging strand as short stretches (100-200 bp), in the correct 5 '- 3' direction. These stretches are known as Okazaki fragments.

Front 31

Okazaki fragments

Back 31

Front 32

Semiconservative- One strand of a daughter chromosome is newly synthesized, the other template strand is inherited intact from the parental chromosome.

Back 32

Front 33

origin of replication
(ori)

Back 33

Sequences where DNA replication begins. Factors recognize and bind the specific ori sequence, unwind its DNA, and attract the components of the replication apparatus, which moves on down the chromosome replicating DNA as it goes.

Front 34

How many ORIs do prokaryotes and eukaryotes have?

Back 34

Proks 1, Euks multiple; this is how eukaryotic DNA replication takes only a little more time than prokaryotic, even though our genome is 1000s of times larger

Front 35

How can eukaryotic DNA replication be bidirectional?

Back 35

two replication apparatuses actually assemble around the ori and move in opposite directions

Front 36

replicon- region of a eukaryotic chromosome that is replicated as a unit, from one central ori.

Back 36

Replication begins at the ori in the center of a replicon, and extends in both directions until it reaches the end of an adjacent replicon. Because there are multiple replicons, different regions of the genome are replicated simultaneously.

Front 37

What is the average length of our replicons? (BPs)

Back 37

about 200 kb. This is the approximate length of DNA loops anchored to scaffold proteins, suggesting that these proteins may be involved in defining replicon length.

Front 38

helicase

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-factor which unwinds DNA
-binds to an ori where it opens the double helix so DNA polymerases can replicate the strands.
-part of replication apparatus

Front 39

single stranded binding proteins (SSB)

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Factors that stabilize single stranded DNA by preventing it from winding back into a double helix.

Front 40

DNA replication apparatus

Back 40

-cluster of factors assembles around helicase to replicate DNA
-Two apparatuses assemble, one on each side of the ori, and each moves in opposite directions, replicating DNA as they go

Front 41

What factors are part of the DNA replication apparatus?

Back 41

-helicase
-Pol δ
-Pol α
-β clamp
-primase

Front 42

Are SSBs part of the DNA replication apparatus?

Back 42

nope

Front 43

β clamp

Back 43

ring like protein that wraps around DNA to stabilize the association of the replication apparatus

Front 44

β clamp is required for Pol __ processivity.

Back 44

pol δ; Without the clamp, Pol δ only replicates short oligonucleotides (< 200 bp). With the clamp, stretches greater than 100 kb are produced.

Front 45

Pol __ is the enzyme which replicates the leading strand in the ____ direction.

Back 45

Pol δ, 5'-3'; It reads the template one base at a time, incorporating complimentary nucleotides, and ligating their 5' phosphate to the 3' OH of the growing leading strand.

Front 46

As helicase unwinds DNA, single strand binding proteins keep the lagging strand template unwound.

Back 46

Front 47

primase

Back 47

enzyme binds the unwound lagging strand template and transcribes a short stretch of RNA (<15 bp). This RNA serves as a primer, providing the 3' OH group required by Pol α.

Front 48

DNA polymerase α (Pol α)

Back 48

enzyme uses the RNA primer to synthesize an Okazaki fragment of the lagging strand.

Front 49

Does Pol α have proofreading capability?

Back 49

no, suprisingly

Front 50

Why is the lagging strand held in a loop?

Back 50

as Pol α synthesizes one Okazaki fragment, helicase continues unwinding single stranded DNA for the next fragment. Therefore, the lagging strand is held in a loop.

Front 51

What happens to the okazaki fragments?

Back 51

Pol α continues replicating the Okazaki fragment until it reaches the primer at the end of the previous fragment. The lagging strand is then released, primase makes the next primer at the end of the new single stranded region, and the process is repeated.

Front 52

Removal of RNA Primers from the Lagging Strand

Back 52

After the replication apparatus passes, it leaves a lagging strand full of RNA primers which must be removed. This involves three separate enzymes:
-RNAase
-DNA Pol β
-DNA ligase

Front 53

RNAase

Back 53

This enzyme digests any RNA.
In replication, it serves to remove the lagging strand primers.

Front 54

DNA polymerase β (Pol β)

Back 54

fills in gaps in DNA, about 20 bp long.
In replication, it fills in the gaps left after the RNA primers are removed. Note, DNA polymerases can only add nucleotides; they cannot link DNA fragments. Therefore, Pol β leaves nicks in the DNA.

Front 55

DNA ligase

Back 55

enzyme binds any free 3’ hydroxyls and 5 phosphates of DNA.
In replication, it seals the nicks between the Okazaki fragments left by Pol β.

Front 56

topoisomerase

Back 56

A consequence of unwinding by helicase is that supercoiling increases. Topoisomerases serve to restore the DNA to the proper level of supercoiling.

Front 57

What happens to the overhang left on the end of the chromosome when the last RNA primer is removed?

Back 57

Telomerase fills in the gap; it's a reverse transcriptase which extends the length of telomeres.

Front 58

What would happen without telomerase?

Back 58

If this dilemma were not resolved, then every time dividing cells replicated their DNA, the lagging strands would loss a little bit of telomeric sequence, until their telomeres were completely whittled away. This would destabilize the chromosome.

Front 59

What does telomerase have to do with aging?

Back 59

-Telomerase activity decreases with age, as does the length of our telomeres.
-Primary cell culture lines divide a limited number of times & then die. This is believed to be because they have no telomerase activity.
-In immortalized cell culture lines telomerase is activated, so they can divide indefinitely.

Front 60

What does telomerase have to do with cancer?

Back 60

Telomerase is usually activated in cancer cells, and this is believed to contribute to their immortalization