Lecture 4 - DNA Replication

Question 1

What direction is DNA synthesized?

Answer 1

elongation occurs at the 3’ OH group. template strand is read from 3’ to 5’ while elongation is 5’ to 3’

Question 2

What are the 3 different possible models for DNA synthesis?

Answer 2

continuous - synthesized at the same time semidiscontinuous (how DNA actually replicates) - one strand continuously made, while the other strand is made at intervals discontinuous - strands are made at intervals

Question 3

How is DNA synthesized?

Answer 3

leading strand - continuous; lagging strand - made of okazaki fragments

Question 4

what is DNA ligase?

Answer 4

DNA ligase is an enzyme that can repair nicks in the DNA if there is a 5’ phosphate and a 3’ OH group free

Question 5

what is the primer made of that is needed for DNA synthesis?

Answer 5

RNA

Question 6

How is the RNA primer removed?

Answer 6

DNA Polymerase 1 removes RNA primer and puts DNA there

Question 7

what steps are required for DNA replication?

Answer 7

unwind DNA, relax or add DNA supercoils, make primer, make DNA in a 5’ to 3’ direction, proofread the DNA made, repair gaps, remove primer, seal nicks

Question 8

what regulates initiation in DNA replication?

Answer 8

methylation

Question 9

what happens in elongation?

Answer 9

creation of leading and lagging strands

Question 10

what does initiation require?

Answer 10

requires at least 10 proteins, starts in the sequence OriC, needs two sequences: DnaA binding sites (containing I sites and R sites - 5 repeats of 9bp sequences) and the DUE or DNA unwinding element which is rich in A-T base pairs

Question 11

How does initiation work specifically?

Answer 11

1. 8 DnaA bound to ATP creates helical complex that include the I and R sites of the OriC = this creates supercoiling and strains the A-T (DUE) regions to separate the strands along with other proteins (HU)
2. DnaC along with ATP loads DnaB (helicase) onto separated strands = one DnaB on each strand
3. DnaC hydrolyzes ATP and leaves DnaB around each strand

Question 12

How does elongation work specifically up to the beginning synthesis of lagging and leading strands?

Answer 12

1. Parent DNA is unwound by DnaB
2. topological stress caused by unwinding is relieved by topoisomerases
3. each strand is separated by SSB or single stranded binding proteins
4. synthesis is different for lagging and leading strands

Question 13

what are SSB or single stranded binding proteins?

Answer 13

SSB are proteins that separate the DNA strands so that they dont re-anneal

Question 14

what is DnaB?

Answer 14

DnaB is also known as helicase and is used for unwinding the DNA

Question 15

what does RNA primase do?

Answer 15

RNA primase is used to start DNA replication by priming DNA with RNA, gives the DNA Pol 3 a place to start elongation.

Question 16

Describe the structure of DNA polymerase III

Answer 16

2 core domains of alpha, epsilon, and theta subunits (is a dimer). core domains are linked by clamp loader complex. core domains interact with each other to increase processivity of the complex meaning that it stays on the DNA longer

Question 17

how is the leading strand (3’) synthesized?

Answer 17

1. DnaG (primase) interacts with DnaB to form the primosome - primase makes RNA primer at origin *primer is made in opposite direction to movement of helicase 3’ to 5’

Question 18

how is the lagging strand (5’) made?

Answer 18

utilizes okazaki fragments

1. RNA primer is made by primase
2. extended by DNA Pol 3
3. coordination with leading strand synthesis since DNA Pol 3 is a dimer
4. DNA Pol 1 replaces the RNA primer
5. DNA ligase seals up the nicks

Question 19

what happens during termination?

Answer 19

1. two replication forks meet at terminus region which contains copies of 20bp sequences of Ter
2. sequences enter this, but can’t escape
3. Tus (terminus utilization substance) binds to Ter to make Tus-ter (can only stop replication fork going in one direction)
4. Tus-ter stops both replication forks
5. remaining catenanes (interlocking molecular structures) are separated by topoisomerase 4 (type 2 topoisomerase)

Question 20

what is hemimethylated?

Answer 20

hemimethylated means that the DNA duplex is half methylated. This happens when DNA replication occurs, the daughter strands don’t have methylation while the parents do. Origin of replication is only recognized if both strands are methylated = regulation of initiation.

Question 21

what is Dam?

Answer 21

DNA adenine methylase - it methylates the amino group at C6 of A at GATC sequences; has high affinity for the protein SeqA

Question 22

how does methylation regulated initiation?

Answer 22

hemimethylated oriC sequences interact with plasma membrane using the protein SeqA. SeqA dissociates and oriC sequences are released from membrane. then Dam methylates DNA to allow DnaA to bind and start initiation

Question 23

what is the function of 3’ to 5’ exonuclease DNA polymerase?

Answer 23

when incorrect dNMP is added, the exonuclease removes the mismatch nucleotide and gives polymerase another chance at adding the right nucleotide (not a reverse polymerization) this is called proofreading; low error rate

Question 24

what is proofreading?

Answer 24

not reverse polymerization; it does not involve a pyrophosphate and the mismatch nucleotide is released as a dNMP; also occurs at a different site from polymerization

Question 25

When does replication happen in eukary cells?

Answer 25

during the cell cycle

Question 26

how is eukary DNA organized?

Answer 26

organized into more complex structures with histones to form chromatin

Question 27

how does eukary replication work?

Answer 27

it happens slowly about 50 nucleotides/sec. happens at different origins. 14 different polymerases

Question 28

what is the end problem for chromosomes?

Answer 28

the lagging strand leaves behind a single stranded DNA when the RNA primer is removed. this gap can’t be filled in because DNA Pol 3 has no free 3’ OH groups to extend from.

Question 29

What happens to the DNA at the end of the chromosome after replication?

Answer 29

the extreme parts of the DNA will be lost and won’t be present in the daughter strands. Future rounds of replication will make it shorter. not a problem in circular DNA

Question 30

what are telomeres?

Answer 30

junk DNA at the end of chromosomes that don’t code for anything important; repeated sequences of 6bp added by telomerases

Question 31

what is a telomerase?

Answer 31

telomerase is an enzyme that carries its own template strand in the form of noncoding RNA and can extend telomeres

Question 32

What RNA does the telomerase contain?

Answer 32

a 9bp sequence that is 1.5 times repeat nucleotides of the telomere which is complementary to it

Question 33

how does telomerase extend the telomeres?

Answer 33

1. telomerase binds to telomere sequence
2. it extends the 3’ single stranded end of the DNA using internal template using dNTPs
3. 6 nucleotides are added at a time
4. RNA primer is added to the end then DNA Pol extends it
5. nick is sealed by ligase
6. RNA primer is removed but there is still DNA single strand
7. specialized telomere DNA proteins bind to it to prevent recombination and degration of DNA

Question 34

all eukary cells have telomeres, but do they all have telomerase activity?

Answer 34

No, somatic cells lack telomerase activity. Germ cells have this ability and cancer cells too.

Question 35

what happens when a cell’s telomeres get too short?

Answer 35

apoptosis

Question 36

what is the hayflick limit?

Answer 36

limit of the amount of times a cell can safely divide without accruing to many mutations.