Chapter 6 DNA replication, Repair and Recombination - Multiple replication origin shortens replication time
- Once initiator proteins open the helix, other proteins bind to it
- Duplication of DNA is called DNA replication
- DNA replication occurs before a cell divides so two identical Two replication forks form at each replication origin
daughter cells are produced
- In the process of replication, DNA molecules make a Y shape, called
DNA replication the replication fork
- Two replication forks form at each origin and moves along the DNA
- A cell must replicate with extreme accuracy
and opens the helix
- DNA replicates at a rate of 1000 nucleotides per second
- Each strand is used to make a new daughter strand
- Chemical, radiation and reactive molecules cause damage to DNA,
- Replication forks move away from the origin sites in opposite
so it requires repair
direction, that’s why in eukaryotes and bacteria it is called
Base pairing enables DNA replication bidirectional
- Replication forks move fast, about 1000 nucleotide pair per seconds
- In a double helix, each strand is complimentary to the other so each in bacteria and 100 nucleotide pairs in eukaryotes
strand can serve as a template for the synthesis of new strand - The slow rate of movement in human is due to the complex
- Billions of nucleotide pairs are replicated before each cell divides chromatin structure of eukaryotic chromosome
- DNA replication is carried out by a cluster of proteins called the
replication machine
- DNA replication produces two complete helices from one helix
- Semi conservative replication is when both new helices have one
strand of parental template strand and a new strand
DNA synthesis begins at replication origins
- DNA double helices are very stable and the hydrogen bonds keeping
the two strands together can be broken when the temperature is
close to boiling
- Initiator proteins bind to specific site in DNA called the replication
origins where they break the hydrogen bonds and unzip the double
helix
- Hydrogen bonds collectively are very strong but individually are
weak enough for initiator proteins to break one at a time
- In simple cells like bacteria and yeast, replication origins are about DNA polymerase synthesizes DNA using parental strand as a template
100 nucleotide pair
- DNA polymerase catalyzes the addition of nucleotides in the 3OH’
- A-T bonds are easier to break because they have fewer hydrogen
end of the growing nucleotide daughter strand
bonds, so replication origins are rich in A-T base pairs
- The new daughter strand is built complimentary to the parental
- Bacterial circular DNA has a single replication origin
template strand
- Humans have 10000 such origins, 220 origins per chromosome
, - The incoming nucleotides form phosphodiester bonds between the - If a wrong base pair is made, polymerase carries out
5’P end and the growing 3’OH of the DNA proofreading
- The incoming nucleotide is in the form of deoxyribonucleoside - Proofreading takes place simultaneously with DNA synthesis. Before
triphosphate dNTP polymerase adds a new nucleotide, it checks the base pair. If it is
- The pyrophosphate is lost and is further hydrolyzed wrong, the nucleotide and adds the correct one
- The energy needed for the formation of the phosphodiester bond - Proofreading is carried out by nuclease, and it cleaves the
between the nucleotides comes from the hydrolysis of the incoming phosphodiester bond
nucleotide - If DNA was synthesized in the 3’ to 5’ direction, then when
- DNA polymerase does not dissociate from the DNA strand each time proofreading, after a wrong base is removed, it would create a dead
a new nucleotide is added, it moves along the strand adding more end and thus the chain can no longer be elongated
nucleotides
Short strands of RNA act as primers
The Replication Fork Is Asymmetrical
- Short lengths of RNA (about 10 base pairs long) are added to the
- At each replication fork, the two DNA strands run in opposite DNA template provides a 3’ end starting point for the DNA synthesis
direction, one in 3’ to 5’ direction and the other in 5’ to 3’ - Enzyme that synthesizes RNA primers is called primase
- It is observed that DNA is synthesized in both directions, but that is - Primase synthesizes RNA using DNA template
not correct - RNA differs from DNA in the way that its sugar is ribose sugar
- Nucleotides can only be added in the 5’ to 3’ direction - And instead of base pair, T, it has U. U pairs with A
- That’s why, in the “wrong direction” strand, referred to as the - In the leading strand, there is only one RNA primer needed at the
lagging strand is synthesised in fragments origin
- Lagging strand was discontinuously synthesised in the 5’ to 3’ - In the lagging strand, a new primer is made at intervals, so a new
direction and polymerase is moving backwards and dissociates Okazaki fragment is made at each 3’ primer end
every time a small chunk is synthesized - To make a continuous DNA strand on the lagging strand, three other
- the small chunks are called Okazaki fragments and they are enzymes are needed
connected later to make the strand continuous; they are about 200 - These enzymes remove the RNA primer, replace it with DNA and
nucleotides long join the DNA fragments together
- An exonuclease enzyme degrades the RNA
DNA polymerase is self correcting
- Repair polymerase replaces the region with DNA
- DNA polymerase only makes about one error every 10 7 nucleotides - DNA ligase uses ATP to join the 5’ phosphate end with the 3’OH end
- Incorrect base pairings like G-T and A-C can sometimes be formed, of the next fragment
and if not corrected, they result in accumulation of mutations - Primers are not proofread, so when primase enzyme synthesizes
- Polymerase reads the strand in 5’ to 3’ RNA primers, there are several mistakes
- DNA polymerase prevents this by: - But that does not matter because primers are removed anyway
- Monitoring the base pairs carefully between the strand and - So when repair polymerase makes new DNA, it is proof read and
the incoming nucleotide, only if it is the correct base pair, DNA is copied correctly
polymerase catalyzes the reaction
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