Nucleic acids 2- DNA replication and mitosis Flashcards
What is the function of DNA helicase
The DNA helix is very stable and has to be unwound before replication can occur. This is done by a DNA helicase, an enzyme that uses ATP as source of energy to break hydrogen bonds between base pairs.
How is new DNA synthesised
New DNA is synthesised by
enzymes called DNA polymerases. DNA polymerases add nucleotides to the 3’ end of a growing chain.
What do DNA polymerases require for DNA synthesis
A template strand
An obligonucleotide primer- they cannot synthesise DNA from scratch- need a start point
deoxynucleotide tri-phosphate- hydrolysis of anhydride bonds provides energy for DNA synthesis.
Describe how DNA polymerase works
DNA polymerases add dNTPs to the 3’ end of a DNA molecule.
DNA (and RNA) synthesis occurs in 5’ to 3’ direction.
Energy is released by hydrolysis of the triphosphate. This drives the reaction.
A free 3’ hydoxyl group is required- to form the phosphodiester links.
How do nucleoside analogues act as chain terminators
They do not have a hydroxyl group on the 3’ carbon to form phosphodiester bonds- hence stopping the action of DNA polymerase.
What is the site where DNA replication begins called
Replication origin
What is the point of DNA synthesis called
Replication fork- it continuously moves in one direction during the process.
Describe the direction in which new DNA strands and the situation that arises from this characteristic.
The replication fork is asymmetric. Both strands are synthesised in a 5’-3’ direction. The leading strand is synthesised continuously, whereas the lagging strand is synthesised in short pieces termed Okazaki fragments.
Describe the action of RNA primers
RNA primes the synthesis of new DNA. A specialised RNA polymerase called DNA primase synthesises a short RNA fragment (~ 10 nucleotides). The RNA primer is only transient and removed at a later stage of replication.
For the synthesis pf the leading strand, an RNA primer is needed only to start replication at a replication origin.
Describe the synthesis of the lagging strand.
New RNA primer made by RNA primase. DNA polymerase adds to RNA primer creating new okazaki fragments- finishes fragment up until previous Okazaki fragment. Old RNA primer erased and replaced by DNA- DNA ligase then joins the okazaki fragments together- using ATP- making the strand continuous.
How are RNA primers removed
A special ribonuclease removes RNA primer using a 5’ to 3’ exonuclease activity.
A repair DNA polymerase then replaces RNA with DNA.
What is the purpose of the sliding clamp
To ensure that the DNA polymerase does not ‘fall off’ the DNA double helix
How does DNA polymerase proof read DNA
Before a new nucleotide is added, the previous nucleotide is checked for correct base-pairing.
Any incorrect bases are removed by 3’ to 5’ exonuclease activity of DNA polymerase. A new, correct nucleotide is then added.
Why is the proof reading mechanism important.
The high fidelity of DNA replication requires a proof-reading mechanism to ensure no mistakes are made. Mutations (changes in DNA sequence) are very dangerous to the organism. Any errors in replication cannot be repaired.
Describe the replication of the E.coli chromosome
In E.coli, replication starts at a unique origin, OriC. Two replication forks proceed simultaneously in opposite directions. The two forks meet at the other side of the circular chromosome.
Describe the replication of the eukaryotic genome
Eukaryotic chromosomes are linear and very long. Multiple replication origins are distributed at intervals of about 100 kilobase pairs- for fast replication. Each replication origin gives bi-directional replication forks. Replication is finished when all the forks have met.
Describe the stages of the cell cycle
M phase: Mitosis; cell division; 1hr
G1 phase: Gap phase 1 (prior to DNA syntesis); 10 hrs
Makes proteins and RNA
S phase: period of DNA synthesis (replication); 9hrs
G2 phase: Gap phase 2 (between DNA synthesis and mitosis); 4 hrs
Grows, makes more proteins.
G0: cells which have stopped dividing entered senescence.
Describe how the chromosomes appear in the cell cycle
G1: DNA of each chromosome present as a single linear double helix of DNA
S phase: the DNA is replicated
G2: each chromosome has two identical sister chromatids
Mitosis: the two chromatids separate to the daughter cells
Describe the stages of mitosis
interphase- chromosomes not visible
Late prophase- chromosomes condense and become visible- each chromosome has two sister chromatids- nucleus disintegrates.
Metaphase- Condensed chromosomes,
aligned on central plane of spindle
Anaphase- Sister chromatids separate & are pulled to spindle poles
telophase- Sister chromatids move to opposite poles of spindle
Cytokinesis- Two daughter cells- cleavage of cytoplasm
Interphase (G1)- condensation process reversed.
