Replication of DNA Flashcards
1) Describe the initiation of DNA replication in prokaryotes
- Proteins interact at the origin of replication on DNA
- Double helix is opened up with aid of helicase enzymes (initiator proteins) and 2 replication forks are formed, exposing single stranded templates for DNA synthesis
2) How is the rest of the DNA strand affected when helicase opens up the double helix?
- When opened, torsion is sent down the double helix (twisting)
3) Which enzymes must act on the supercoiled DNA strands before initiation and what do they do?
- Topoisomerase enzymes which uncoil the supercoiled DNA strands, ahead of replication
> Type I relaxes the supercoiled DNA
> Type I (gyrase) cuts and releases the DNA strand to relieve supercoiling (loosens twist -> straightens)
4) Which direction is new DNA synthesized?
From the 5’ -> 3’ end
5) What is the leading strand and the lagging strand in DNA replication?
- Leading strand: strand of DNA from which the new DNA strand is synthesized continuously
- Lagging strand: the other strand of DNA from which the new strand is synthesized as a series of short [Okazaki] fragments (discontinuously)
6) Why can the lagging strand not be used to synthesize a new DNA strand continuously? [Polarity problem]
- This would be synthesized from 3’ to 5’ end which contradicts the 5’ -> 3’ end rule
- Replication machinery must move from the 5’ -> 3’ end, as the parent strands are unwound
7) How is the lagging strand still able to produce a new strand of DNA despite initially running in the 3’ -> 5’ direction?
- Lagging strand template is looped to allow synthesis (so is running in the opposite direction to itself, parallel)
- DNA polymerase III makes new DNA on both strands simultaneously and moves in the same direction as the replication fork
- Loop enlarges as RNA primers move along the strand
8) Name the 3 enzymes involved in the initiation stages of DNA replication
- Helicase - unwind DNA strands at replication fork
- Topoisomerases - release supercoils in DNA
- Single strand binding proteins - stabilise single stranded DNA in separate strands
9) Which enzyme attaches DNA polymerase III firmly to the DNA strand?
- beta protein (sliding clamp)
10) What are the functions of DNA polymerase III and primase in DNA replication of the leading and lagging strand?
- Leading : requires primase(makes RNA primer to begin DNA synthesis) only at the beginning of the synthesis of the new strand
- Lagging: Requires primase at the beginning of each new Okazaki fragment (RNA primers needed for each)
- Both strands need DNA Polymerase III to synthesize the new DNA strand
11) What are the functions of DNA polymerase I and DNA ligase in DNA replication of the leading and lagging strand?
- DNA polymerase I : transfers complementary deoxy nucleotides to the leading and lagging strand
- removes the nucleotides of the RNA primer and adds deoxynucleotides to fill the gap with DNA
- Ligase : joins discontinuous Okazaki DNA fragments by making a phosphodiester bond (in lagging strand) and joins nucleotides with the same bond (in leading)
12) How can mutations be created in the new DNA strand?
- Incorporation of incorrect nucleotides into the new DNA chain
13) Which 2 enzymes have a proof-reading ability and how is mutated DNA repaired?
- Polymerase I and III can detect incorrect base pairs
- 3’ -> 5’ (reverse) exonuclease activity removes the incorrect base and polymerase inserts the correct base instead
- Errors that escape this proof reading are also repaired in E.Coli
[Another protein group can removes a section of newly formed DNA and PolyIII resynthesizes the new strand]
14) What does it mean DNA has a high ‘fidelity’ and why is this so important?
- low error rate
- as consequences of a single base mutation are serious
( mutated gene -> abnormal protein -> loss of important function/gain of toxic function (e.g.cancers) )
15) Give 8 ways in which DNA can be damaged (and may be repaired by DNA repair complexes)
- UV light : thymine dimer formation
- Ionising radition : strand breaks
- Nitrous acid : cytosine converted to uracil
- Alkylating agents : guanine modification (GC->AT)
- Free radicals : strand breaks, base modification
- Bulky chemicals : distorts double helix
- Spontaneous : cytosine -> uracil, CG becomes T, loss of purines
- Carcinogenic chemicals (smoke) -> various base modifications
