DNA Replication Flashcards
What is semi-conservative replication?
- Both strands of double helix separate, each acts as a template to direct the synthesis of a complementary daughter strand
- H-bonds form between complementary bases of one old strand and one new strand to form a complete DNA molecule
- DNA molecule in the daughter cell is a hybrid of one old strand and one new strand.
What are the characteristics of semi-conservative replication?
- Rapid - 6 billion base pairs are copied in a few hours in human cells
- Accurate - error rate of one in a billion nucleotide
What are the required factors in DNA replication?
- Template strand (from parental DNA)
- RNA primer
- Enzymes:
- Helicase/ Topoisomerase
- DNA polymerase III
- DNA polymerase I
- Primase
- DNA ligase
- Single-stranded DNA binding proteins
- Gyrase/ Type II topoisomerase
- Precursor deoxyribonucleotides (dNTP, N=A, T, C, G)
- ATP (energy source)
- Inorganic ions (eg Mg2+)
What are the steps in DNA replication?
- Unwinding of DNA by helicase
- Binding of single-stranded DNA binding proteins
- Synthesis of RNA primer
- Elongation + synthesis of daughter strand
- Replacing RNA primer with DNA
- Closing nicks in DNA
- Rewinding of DNA helix
What happens during unwinding of DNA?
- Helicase recognises and binds to the origin of replication, causing the DNA molecule to unwind and unzip at the origin of replication.
- Hydrogen bonds between the bases are broken and the DNA strands are separated.
- Two replication forks spread in both directions, creating a replication bubble, exposing two parental strands as template strands for replication to take place.
**Helicase requires energy from ATP hydrolysis
**Eukaryotes have numerous origins of replication and many replication bubbles form along the length of the DNA
What happens during the binding of the single-stranded DNA binding proteins?
- Single-stranded DNA binding proteins bind to the separated strands of parental DNA
- Stabilizes the single-stranded DNA, keep strands apart, prevent it from denaturing before DNA replication can occur
- unwound region serve as a template for DNA replication
What happens during the synthesis of RNA primer?
- Primase attaches to DNA template strand, synthesises a RNA primer (consist of about 10 ribonucleotides) that is complementary to the template
Why is there a need for RNA primers?
- For the addition of new deoxyribonucleotides
- DNA polymerase III can only add new deoxyribonucleotides to the 3’ OH group of the sugar of the previous nucleotide in an existing chain, while primase does not require a primer to initiate polynucleotide synthesis
- Hence, RNA primers are the beginnings of all new DNA chains, since DNA polymerase III cannot add deoxyribonucleotides directly to the bare parent template
What happens during elongation and synthesis of daughter strand?
- DNA polymerase III recognises the bases on the template strand, select free deoxyribonucleotides that are complementary to those on the parental strand
- DNA polymerase III catalyses the formation of a phosphodiester bond.
- It also proofreads each nucleotide against its template, removing the mismatched deoxyribonucleotide and inserting the correct one before proceeding with polymerisation.
**parental strand is read in 3’ -> 5’ direction while new daughter strand is synthesized from 5’ to 3’ direction
What happens during the replacement of RNA primer with DNA?
- RNA primer is hydrolysis by DNA polymerase I
- Removal of RNA leaves substantial gaps between DNA fragments
- DNA polymerase I fills in these gaps by adding complementary deoxyribonucleotides to the 3’ OH group of the sugar of the previous nucleotide in DNA fragment upstream
** There is still a nick which DNA polymerase I cannot seal.
What happens during the closing of nicks in DNA?
- DNA ligase catalyses the formation of a phosphodiester bond between 2 nucleotides, sealing the nicks left behind by DNA polymerase I.
- DNA ligase requires a free hydroxyl group at the 3’ end of one DNA chain and a free phosphate group at the 5’ end of another DNA chain —> joining the sugar-phosphate backbone of Okazaki g=fragments to form one strand
What is the difference between nicks and gaps?
- Gap: missing nucleotides
2. Nick: missing phosphodiester bond
What happens during the rewinding of DNA helix?
- Both parental and daughter strands rewind into double helix
- The process is semi-conservative since each resultant double helix consists of one parental strand and one new daughter strand.
What is a leading strand?
- Synthesised continuously from 5’ to 3’ in the direction towards the helicase/ replication fork
- DNA polymerase III nestles in the replication fork on template strand, and continuously adds nucleotides to the complementary strand.
What is a lagging strand?
- Synthesised discontinuously in the form of Okazaki fragments, which is initiate by a RNA primer before the addition of deoxyribonucleotides.
**DNA polymerase III: attaches to lagging strand template, synthesises a short segment of DNA (other segments of the lagging strand are made in a similar way)
**DNA polymerase I: plays an important role in removing numerous RNA primers along lagging strand, replacing them with DNA sequences
**DNA ligase: plays an important role in sealing the numerous nicks along the lagging strand
