chapter 5: DNA replication, end-replication problem & telomeres

Question 2

the first replication in the 14N medium produced a band of hybrid 14N-15N DNA molecules of intermediate density:

• the parental 15N-15N molecule unwinds, ________ and ______ , and each 15N strand acts as a ______ for the synthesis of a new ________
• ______ containing 14N are added via complementary base pairing with the template strand to form the new daughter strand
• each daughter _______ consists of. one 15N parental strand which is ‘heavy’ and one 14N daughter strand which is ‘ light;
• hence, this results in hybrid 14N-15N DNA molecules which are of ________ ________

Answer 2

• the parental 15N-15N molecule unwinds, unzips and separate , and each 15N strand acts as a template for the synthesis of a new daughter strand
• deoxyribonucleotides containing 14N are added via complementary base pairing with the template strand to form the new daughter strand
• each daughter DNA molecule consists of one 15N parental strand which is ‘heavy’ and one 14N daughter strand which is ‘ light;
• hence, this results in hybrid 14N-15N DNA molecules which are of intermediate density

Question 3

the second replication in 14N medium produced a band of hydrid 14N-15N DNA molecules and a band of 14N-14N DNA molecules

• the parental 14N-15N molecule unwinds, ________ and ______ , and each 15N strand acts as a ______ for the synthesis of a new ________
• ______ containing 14N are added via complementary base pairing with the template strand to form the new daughter strand
• half of the daughter DNA molecules would consist of one 15N parental strand and one 14N daughter strand each, resulting in 14N-15N DNA molecules of intermediate density
• The half would consist of one 14 N daughter strand and one 14N parental strand each, resulting in 14N-14N DNA molecules of light density

Answer 3

• the parental 14N-15N molecule unwinds, unzips and separates , and each 15N strand acts as a template for the synthesis of a new daughter strand
• deoxyribonucleotides containing 14N are added via complementary base pairing with the template strand to form the new daughter strand
• half of the daughter DNA molecules would consist of one 15N parental strand and one 14N daughter strand each, resulting in 14N-15N DNA molecules of intermediate density
• The half would consist of one 14 N daughter strand and one 14N parental strand each, resulting in 14N-14N DNA molecules of light density

Question 4

INITIATION:
what are origins of replication and how is it different in prokaryotes and eukaryotes?

Answer 4

• DNA replication begins at specific sites called the origins of replication, where two parental DNA strands separate to form a replication bubble
• this site is usually rich in adenine and thymine

in prokaryotic cells:
- only one chromosome is present
- this chromosome is in the form of a circular DNA molecules and with a single origin of replication

in eukaryotic cells:
- the genome and DNA molecules are comparatively larger in size
- eukaryotic chromosomes are linear
- many origins of replication are present
- multiple replication bubbles form and eventually fuse to give two complete daughter DNA strands, thus increasing the rate at which DNA replication occurs

Question 5

INITIATION:
describe strand separation

Answer 5

• after DNA is unwound from the histone proteins, the enzyme helical recognises and binds to DNA at the origin of replication, and unwinds and unzips the DNA molecule by breaking the weak hydrogen bonds between the bases
• this separates the parental strands, exposing the template for DNA replication
• helicase is ATP dependent, its activity requires energy to break strands apart
• replication of DNA then proceeds in both directions from the origin of replication until the entire molecule is copied
• unwinding produces a replication bubble which contains two replication forks
• the separated strands of parental DNA are unstable and have the tendency to reform the DNA double helix
• single-strand DNA-binding proteins thus bind to separated strands of parental DNA, which stabilised the unpaired DNA strands and keeps the strands apart,
• while they serve as templates for the synthesis of new complementary DNA strands
• unwinding also causes the helix ahead of the replication fork to rotate, causing further twisting and strain ahead of a replication fork
• topoimerase helps to relieve strain by breaking, swiveling and re-joining DNA strands

Question 6

INITIATION:
describe the process of priming DNA synthesis

Answer 6

• before DNA synthesis can begin, there must be small pre-existing primers to start the addition of new nucleotides
• the enzyme primase catalyses the synthesis of a short RNA chain of around ten ribonucleotides called an RNA primer that is complementary to the 3’ end of the parental DNA template
• the enzyme which is directly involved in the synthesis of the new DNA strand is DNA polymerase
• a primer is required because DNA polymerase cannot initiate the synthesis of a polynucleotide strand
• DNA polymerase cannot only add deoxyribonucleotides to a free 3’ OH end of a pre-existing strand that is already base paired with the template strand
• this is due to the active site specificity of DNA polymerase which is only complementary in 3D conformation to a free 3’ OH group of a pre-existing chain base paired to the template Adna
• this is also the reason why DNA replication occurs in the 5’ to 3’ direction
• the RNA primer thus provides a 3’ OH end available for DNA polymerase
• it is base paired to the complementary DNA template strand and is anti parallel to the template strand

Question 7

describe elongation: synthesis of new DNA strands

Answer 7

• DNA elongation of the daughter strand only occurs in the 5’ to 3’ direction
• this means that nucleotides are added to the free 3’ OH end of a growing DNA strands
• each parental strand acts as a template to determine the order of the bases to be added to the new daughter strand through complementary base pairing via hydrogen bond formation between the bases
• A pairs with T, and G pairs with C
  -DNA polymerase catalyses the addition of DNA nucleotides and formation of phosphodiester bonds between adjacent DNA nucleotides, in the 5’ to 3’ direction
• as DNA polymerase moves along the template, part of the enzyme ‘proofreads’ the previous region
• this is to check if proper base pairing has taken place between the bases
• if a wrongly paired deoxyribonucleotide was added, this would be swiftly removed by the enzyme and replaced with the correct DNA nucleotide
• a different DNA polymerase then removed the RNA primer and replaces it with DNA nucleotides

Question 8

anti-parallel elongation of DNA strands

• since the parental strands are _______, the two daughter strands are synthesised in ________ _______ with respect to each other because
• _________ can only add deoxyribonucleotides to a free 3’ OH end of a _______ _________. hence elongation can only occur in the ___ to ___ direction
• the parental template DNA strands are antiparallel to each other
• one of the elongating strands is known as the ______ strand while the other is known as the _____ strand

Answer 8

since the parental strands are antiparallel, the two daughter strands are synthesised in opposite directions with respect to each other because
- DNA polymerase** can only add deoxyribonucleotides to a free 3’ OH end of a pre-existing strand hence elongation can only occur in the ** 5’ to 3’ ** direction
- the parental template DNA strands are antiparallel to each other
- one of the elongating strands is known as the leading strand while the other is known as the lagging strand

Question 9

ELONGATION:
describe the process of the synthesis of the leading strand

Answer 9

• along one of the template strands, DNA polymerase adds DNA nucleotides continuously in the 5’ to 3’ direction as the replication fork unwinds
• the new strand is synthesised continuously as a single polymer towards the replication fork
• the DNA strand made by this mechanism is called the leading strand

Question 10

ELONGATION:
describe the process of the synthesis of a lagging strand:

Answer 10

1. as the replication fork unwinds, some template is exposed
   - primase adds ribonucleotides that are complementary to the 3’ end of the template and catalyses the formation of phosphodiester bonds between adjacent RNA nucleotides to form an RNA primer
2. next, DNA polymerase adds DNA nucleotides to the 3’ OH of the RNA primer, forming the first okazaki fragment in the 5’ to 3’ direction
3. as more of the template strand is exposed with the unwinding of DNA at the replication fork, a second RNA primer is synthesised by primase
   - DNA polymerase then adds DNA nucleotides to the second primer and detaches when it reaches the first primer
4. another DNA polymerase then removes the RNA primer and replaces it with DNA nucleotides
5. DNA ligase next forms a phosphodiester bond between free OH group at 3’ end of each new okazaki fragment to the phosphate group at the 5’ end of the growing chain

Question 11

synthesis of the lagging strand:

• the lagging strand is synthesised _______ and is produced as a series of short ______ of DNA nucleotides called _______ _______
• each okazaki fragment is synthesised in the __ to __ direction by DNA polymerase, and eventually joins up with the other fragments, forming a _____ DNA strand
• the direction of synthesis of each okazaki fragment is _____ from the replication fork
• the DNA strand synthesised in this direction is called the lagging strand

Answer 11

• the lagging strand is synthesised discontinuously and is produced as a series of short segments of DNA nucleotides called ** okazaki fragments**
• each okazaki fragment is synthesised in the 5’ to 3’ direction by DNA polymerase, and eventually joins up with the other fragments, forming a continuous DNA strand
• the direction of synthesis of each okazaki fragment is away from the replication fork
• the DNA strand synthesised in this direction is called the lagging strand

Question 12

describe the process of termination

Answer 12

• the termination of replication occurs when two replication forks meet each other
• the complementary parental and daughter DNA strands rewind into a double helix
• the process is semi-conservative since each new DNA molecule consists of one parental DNA strand and one newly synthesised daughter DNA strand
• mismatched nucleotides sometime evade proof reading mechanism by DNA polymerase
• after replication, DNA repair enzymes can recognise mismatched nucleotides, remove the nucleotides, and replaces it them with the correct nucleotides based on complementary base pairing rule
• this mechanism is also responsible for the repair of DNA that might have been damaged by chemicals and radiation

Question 13

what does the end replication problem result in?

Answer 13

• the 3’ ends of the template DNA strand not being replicated
• which also means the 5’ ends of the newly synthesised daughter strands are shorter relative to that of the previous generation

Question 14

how does the end replication problem occur?

Answer 14

• when the lagging strand is synthesised, many okazaki fragments are formed
• each fragment has a primer made by primase
• DNA polymerase removes the primers and fills the gap with complementary deoxyribonucleotides
• however at the 5’ end of the lagging strand, the last primer cannot be replaced with DNA nucleotides because of the lack of an existing 3’OH end of a pre-existing strand
• therefore, complete replication of the 5’ ends of daughter DNA strands cannot occur, and as a result, the new daughter chromosome formed lack a certain length of DNA at each 5’ end
• this causes chromosomes to become shorter with each cell division
• after repeated rounds of replication and cell division, essential genes would be eroded from the ends of the chromosomes

Question 15

what is the structure of telomeres?

Answer 15

• telomeres are found at both ends of a linear chromosome
• telomeres consist of multiple tandem repeats of a short, non-coding DNA sequence, which varies from species to species

Question 16

what are the 5 functions of telomeres?

Answer 16

1. prevents essential genes at the ends of chromosomes from being eroded/ lost with each round of DNA replication
2. prevents chromosomal end-to-end fusion
3. protects chromosomal ends from degradative enzymes
4. causes cellular aging
5. prevents cancer development

Question 17

how does telomeres prevent essential genes at the ends of chromosomes from being eroded with each round of DNA replication?

Answer 17

• the non-coding, multiple repetitive sequences in telomeres serve as a buffer DNA which protects the organism’s genes from being eroded after successive rounds of DNA replication
• instead, telomeric DNA sequence is eroded in place of the organism’s genes, telomeres thus have a ‘ disposable buffer effect’
• this allows for critical proteins encoded by genes located near the ends of the chromosomes to continue to be expressed in the daughter cells despite the shortened chromosomes

Question 18

how do telomeres prevent chromosomal end-to-end fusion?

Answer 18

• The telomere ends loop back and bind with telomere-binding proteins to form a protective telomeric cap.
• This cap stabilizes chromosomes by preventing single-stranded ends from fusing with other chromosomes.
• Without telomeres, chromosome ends may undergo complementary base pairing with other terminal ends, leading to chromosomal fusion or joining of sister chromatids.
• Such mutations activate the cell’s DNA damage response, halting division and leading to cell death.

Question 19

how do telomeres protect chromosomal ends from degradative enzymes?

Answer 19

• the telomeric cap also maintains chromosome stability by preventing the otherwise single-stranded chromosomal ends from being recognised and degraded by cellular exonucleases

Question 20

how does telomeres cause cellular aging?

Answer 20

• Telomeres act as a molecular clock, shortening by 50–200 base pairs with each cell division.
• After 40–60 divisions, when telomeres reach a critical length (the Hayflick limit), cells either stop dividing (replicative senescence) or undergo programmed cell death (apoptosis).
• Replicative senescence refers to a permanent state where cells remain alive but can no longer divide, while apoptosis is the controlled self-destruction of damaged or unnecessary cells.
• This highlights the role of telomeres in regulating cellular aging.

Question 21

how does telomeres prevent cancer development?

Answer 21

• the shortening of telomeres also indirectly prevents the development of cancer
• this is because cells may undergo apoptosis (programmed cell death) when their telomeres have been shortened to a critical length
• apoptosis thus prevents accumulation of mutations, thus preventing cancer development

Question 22

what is the role of telomerase?

Answer 22

• as telomeres shorten, cells age and eventually die
• this is a normal and natural progression for most cells
• in cells such as stem cells that need to divide repeatedly, an enzyme telomerase is expressed
• telomerase catalyses the lengthening of telomeres
• hence, in cells that express telomerase, the end replication problem is overcome as telomeres can be lengthened with each round of division.

Question 23

what is telomerase and what are the different components?

Answer 23

• telomerase is a ribonucleoprotein that functions as a reverse transcriptase

RNA component:
- part of the RNA sequence complementary base pair via hydrogen bonds with the 3’ end of telomere to help attach the telomerase to the DNA
- another part acts as a template for the enzymes to elongate the 3’ end of the telomere by insertion of multiple repeats of deoxyribonucleotides by complementary base pairing

protein component:
- acts as a reverse transcriptase
- the active site is able to catalyses the formation of phosphodiester bonds between deoxyribonucleotides of multiple DNA repeats
- which are added via complementary base pairing with the RNA template at the 3’ end of the telomere by

Question 24

what is telomerase and what are the different components?

Answer 24

• telomerase is a ribonucleoprotein that functions as a reverse transcriptase

RNA component:
- part of the RNA sequence complementary base pair via hydrogen bonds with the 3’ end of telomere to help attach the telomerase to the DNA
- another part acts as a template for the enzymes to elongate the 3’ end of the telomere by insertion of multiple repeats of deoxyribonucleotides by complementary base pairing

protein component:
- acts as a reverse transcriptase
- the active site is able to catalyses the formation of phosphodiester bonds between deoxyribonucleotides of multiple DNA repeats
- which are added via complementary base pairing with the RNA template at the 3’ end of the telomere

Question 25

describe the mode of action of telomerase.

Answer 25

1. telomerase binds to the 3’ overhand of the parental DNA strand at the telomeric region, where the telomere repeat sequence on DNA is complementary to the RNA template on telomerase
   - the 3’ end of the parental strand also binds at the active site of the reverse transcriptase RNA component of telomerase
2. DNA nucleotides are added to the 3’ end of the parental DNA strand by CBP
   - using the RNA component of telomerase to as a template
   - formation of phosphodiester bonds between the newly added DNA nucleotides is then catalyses by the reverse transcriptase RNA component component of telomerase
3. telomeras thus extends the 3’ overhang of the parental DNA strand in the 5’ to 3’ direction
4. after the formation of one repeat sequence at the 3’ of the parental DNA strand, the telomerase transloccates to form another repeat sequence
5. steps 2-4 are repeated and the 3’ end o the parental strand is lengthened
6. DNA replication- not by telomerase:
   - replication of shorter daughter strand is completed by using the extended 3’ overhand as a template, for the synthesis of a complementary daughter strand using primase and DNA polymerase

Question 26

what is telomerase present in?

Answer 26

1. unicellular eukaryotes
   - because cell division is the only mode of reproduction for these organisms
2. stem cells:
   - to ensure that the cells are all blue to undergo continual self-renewal via mitosis
   - for example, telomerase expression is high in embryonic stem cells to sustain enough cell divisions to form an organism
3. cancer cells:
   - where the activity of the telomerase is abnormally turned on
   - telomerase maintains telomeres at a length which allows continued cell divisions to
   - the senescence programme is bypassed, and cells undergo uncontrolled cell divisions to
   - resulting in the eventual formation of a tumour