DNA replication, end-replication problem & telomeres

Question 1

process of DNA replication

what happens during INITIATION (part 1: strand separation ) ?

Answer 1

• once DNA unwounds from the histone proteins, the enzyme helicase 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 them as template strands
• helicase is ATP-dependent
• replication occurs 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 separate strands of parental DNA are unstable and have the tendency reform the double helix
• single-strand DNA-binding proteins thus bind to the seperated strands of DNA
• which stabilises the unpaired DNA strands and keeps them apart, while they serve as template for the synthesis of the 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, swivelling and re-joining DNA strands

Question 2

process of DNA replication

what happens during INITIATION (part 2: priming DNA synthesis)

Answer 2

• before DNA synthesis can begin, there must be small pre-existing primers to start the addition of new neucleotides
• the enzyme primase catalyses the synthesis of a short RNA chain of around 10 ribonucleotides called **RNA primer **that is complementary to the 3’ end of the parental DNA template
• DNA polymerase is the enzyme directly involved in the synthesis of a new DNA strand
• but it cannot initiate the synthesis of a polypeptide strand which is why a primer is needed
• DNA polymerase can only add deoxyribonucleotides to a free 3’ OH 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 complementary in 3D conformation to a free 3’ OH groupof the pre-existing strand
• also the reason why DNA replication occurs in the 5’ to 3’ direction
• RNA primer thus provides a 3’OH end availible for DNA polymerase
• it is base-paired to the commplementary DNA template strand and it is parallel to it

Question 3

process of DNA replicaiton

what happens during ELONGATION?

Answer 3

• DNA elongation fo the daughter strand only occurs from the 5’ to 3’ direction
• new neucleotides are added to the free 3’ OH end of a growing DNA strand
• each parental strand acts as a template to determine the order of bases to be added to the new daughter strand
• through complementary base pairing via hydrogen bond formation between the bases

-DNA polymerase catalyses the addition of DNA nucleotides and the 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 molecule
• a different DNA polymerase then removes the RNA primer and replaces it with DNA nuclotides

Question 4

describe the synthesis of the leading strand.

Answer 4

• 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 is called the leading strand

Question 5

describe the synthesis of the lagging strand

Answer 5

• 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 synthesisted from the 5’ to 3’ direction by DNA polymerase, and eventually joins up with the other fragemnts forming a continuous DNA strand
• it is synthesised away from the replication fork

process:
- 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 the RNA primer

• DNA polymerase then adds deoxyribonucleotides to the 3’ OH end of the RNA primer forming the first okazaki fragment in the 5’ to 3’ direction
• as more of the template strand is exposed by unwinding of DNA at the replicatio fork, a second primer is synthesised by primase
• DNA polymerase then adds DNA nucleotides to the second primer and detaches when it reaches the first primer
• another DNA polymerase then remoces the RNA primer and replaces it with DNA nucleotides
• DNA ligase next forms a phosphodiester bond between the free OH group at the 3’ end of each new okazaki fragment to the phosphate group at 5’ end of the growing chain