DNA replication

Question 1

DNA replication def

Answer 1

mechanism by which the double strands of DNA are replicated - occurs in S phase of cell cycle

Question 2

Location of DNA replication

Answer 2

prokaryotes: cytoplasm
eukaryotes: nucleus

Question 3

How did DNA replication contribute to evolution?

Answer 3

DNA rep occurs with high fidelity with around one mistake per 10 bill nucleotides
-if this mistake is lethal the cell dies via apoptosis
BUT: nonlethal mistakes caused differences (mutations) in DNA rep which led to evolution

Question 4

Semi conservative replication definition

Answer 4

Mechanism of DNA replication by which each strand acts as a template and so that each daughter strand contains 50% of the original DNA and 50%
newly synthesised DNA

!!proposed by watson and crick (but with no experimental evidence)

Question 5

Meselson and Stahl experiment

Answer 5

1. Ecoli bacteria grown in medium containing N15
2. Transferred to medium containing N14
3. Separated by centrifugation for each generation and analysed to see the density of DNA

CONCLUSION:
F1: band in the middle between light and heavy -> this dismissed conservative theory
F2: 2 bands - one hybrid and one light band –> dismissed dispersive theory

HENCE: mechanism for DNA rep is semi conservative

Question 6

What is the main topological problem that DNA rep has to eliminate

Answer 6

The torsional stress caused by the unwinding of the helix which can generate supercoiling (halting replication if unaddressed)

Question 7

Linking number (Lk) def

Answer 7

The number of times a strand of DNA winds around the helical axis when the axis is constrained to lie in a plane

!! Lk cannot change if both strands are intact –> unwinding of the strands causes torsional stress

Question 8

DNA topology def

Answer 8

defines the 3ary structure of DNA

Question 9

General role of topoisomerases

Answer 9

-enzymes regulating topological state of DNA in order to change linking number while preventing supercoiling
-target of drugs
-Type 1 and 2 enzymes
-cutting actions of the enzymes are reversible

Question 10

Type 1 topoisomerase action

Answer 10

Type 1A and 1B:
-don’t use ATP
-can only cut one strand of the DNA helix
1A: uses rearrangement
1B: uses rotation where the 5’ moves circularly
!! changes the Lk by 1

Question 11

Type B topoisomerase action

Answer 11

-use ATP for energy
-can cut both strands of the DNA helix
-use a mix of rearrangement and rotation
!! changes the Lk by 2

Question 12

Origin of replication in prokaryotes (ori)

Answer 12

-prokaryotes have only ONE ori site
-generation of a replication bubble in AT rich areas (because AT has 2 bonds to break vs CG)
-DNA is denatured and strands are separated
-this forms two replication forks which means replication can proceed in opposite directions

Question 13

Origin of replication in eukaryotes

Answer 13

-eukaryotes have MULTIPLE origins of replication
-creation of the replication bubble which separated strands
-creation of forks which allows replication to happen in opposite directions

!! NOTE: eukaryotic replication forks move slower than those in prokaryotes (bcos DNA is more packaged in euk)

Question 14

Initiation in prokaryotes

Answer 14

• DNA A binds to the DUE (DNA unwinding element) which is the AT rich region on the Ori
  -DNA A causes a torsional stress (mechanical NOT enzymatic) that is great enough to separate the strands (!! SOS: strands are unwound but bases are still connected with H-bonding)
  -recruition of DNA C and then DNA B which acts as a helicase to form a ring around one strand and breaks H-bonds to fully separate strands
  -DNA B migrates from the 5’ to 3’ direction using ATP

! SSB proteins are recruited to protect strand from degradation or formation of hairpin structure that could block replication machinery

Question 15

Structure of DNA B helicase

Answer 15

-circular hexamer
-forms a hole in its center and this is important for the strand to pass through it

Question 16

elongation in prokaryotes

Answer 16

-DNAP3 can synthesise phosphodiester bonds only in the 5’ to 3’ direction (leading strand) so this strand is replicated continuously
-the strand in the opposite direction of the fork is the lagging strand and is synthesised in sections (creates Okazaki fragments)
-RNA primase adds primer once to leading strand and constant primer addition to lagging strand.
-these are later removed by DNAP1and replaced with DNA
-DNA ligase links okazaki fragments together

Question 17

structure and function of DNAP in prokaryotes

Answer 17

Bacteria have 5 DNAPs

DNAP3: main one in rep, dimeric holoenzyme, 3 core subunits:

1. CORE POLYMERASE
   -alpha: enzymatic activity
   -epsilon: proofreading
   -theta: stabilises epsilon
2. CLAMP
   -made of 2 beta subunits
3. PALM SITE:
   -palm site is the active site and contains 2 aspartate residues that bind to 2Mg2+ ions
   Mg2+ promotes deprotonation of OH 3’ that attacks phosphate and causes release of pyrophosphate/formation of phosphodiester bond

DNAP1: monomer, removes and replaces RNA primers

DNAP2/4/5: monomers, involved in DNA repair mechanisms

Question 18

processivity of DNAP3 definition

Answer 18

-number of nucleotides added each time the enzyme binds to the template
-DNAP3 has high processivity due to the sliding clamps (2 beta subunits) that prevent it from dissociating from DNA strand

Question 19

Proofreading activity of DNAP3

Answer 19

-in the presence of incorrect base results the base pair is sent be excised:
-DNAP3 active site has a low affinity for single stranded DNA while the epsilon (exonuclease domain) site has a high affinity for it
-ssDNA moves into epsilon site where the base error can be removed

Question 20

General important properties of DNAP3:

Answer 20

1. can only synthesise in the 5’ to 3’ direction
2. requires a primer to add dNTPS

Question 21

what is the replisome

Answer 21

complex formed by the joining of:
DNAP3, DNA B helicase and primase

Question 22

what is the replicone

Answer 22

portion of DNA replicated from the origin to the termination

!! prokaryotes have one replicone, eukaryotes have many (because there are 1 vs many origins of rep)

Question 23

what is the trombone model?

Answer 23

model for elongation that postulates that the lagging strand forms a loop in order for the leading and lagging strands to have protein interaction

Question 24

Key proteins involved in the EUK replication mechanism

Answer 24

-15 DNAPs in eukaryotes
3 key ones:

1. DNAP alpha –> primase for RNA primer synthesis
2. DNAP epsilon –> leading strand synthesis and has proofreading activity
3. DNAP delta –> lagging strand synthesis and proofreading activity

-DNA helicase and ligase are the same
-Fen1 removes RNA primers
-RPA is equivalent to SSB

Question 25

Termination in prokaryotes

Answer 25

• 2 replication forks encounter each other in OPPOSITE directions
  -controlled by Ter sequences (10 exist) which serve as binding sites for Tus proteins (termination utilisation substance)
  -TUS-TER complexes are asymmetrical which means that encounters from BOTH directions create huge kinetic barriers which blocks DNA B helicase and terminates replication

Question 26

What is the problem that arises for termination in eukaryotes?

Answer 26

-every replication removes RNA primers from the lagging strand which induces a SHORTENING in DNA strand length
-only occurs in eukaryotes bcos DNA is linear so it has distinct ends
-this is an issue if the portion of DNA lost codes for fundamental genes

Question 27

Telomeres function and structure

Answer 27

-found at the ends of chromosomes and act as caps –> worn down with each replication
-made of multiple repeating copies of the 6 base sequence:
5’-TTAGGG-3’
-T loop component: avoids the telomeric region from the signal of DNA repair

Question 28

Structure and function of telomerases

Answer 28

-ribonucleoproteins (enzymes) that can extend the telomeres by identifying their ends and using reverse transcriptase

!! Present in high amounts in stem cells and cancer cells