DNA Replication

What are the two principal functions of DNA?

• Template for DNA replication

• Template for RNA replication

Why is DNA termed ‘semi-conservative’?

One new daughter strand is synthesized with each round of DNA replication, but one original strand from the parent molecule is kept

Where does DNA synthesis take place? When does is take place?

• In the nucleus

• It takes place in the S phase (Within interphase)

Within the DNA strand, where does DNA replication take place?

• At replication bubbles or replication forks, such that both strands of the DNA template are replicated at the same time

Which strands are synthesized continuously, which are discontinious?

• The leading strand is synthesized continuously

• The lagging strand is synthesized discontinuously

What are the five steps in the DNA replication process?

1.) Helicases unwind the parental double helix.

2.) Single strand binding proteins stabilize the unwound parental DNA.

3.) The leading strand is synthesized continuously in the 5’ to 3’ direction by DNA polymerase.

4.) The lagging strand is synthesized discontinuously. Primase synthesizes a short RNA primer which is extended by DNA polymerase, forming an okazaki fragment.

5.) After the RNA primer is replaced by DNA (By another DNA polymerase), DNA ligase joins the okazaki fragment to the growing strand.

What is the main function of DNA Polymerase? What enzyme does it need to perform it’s job?

• Enzyme that synthesizes DNA, only synthesizes in the 5’ to 3’ directions

• In order to do so it requires a starter or primer sequence which is synthesized by the enzyme primase

What is the main function of the primase enzyme?

• Primase synthesizes short RNA primers that are complementary to a single-stranded DNA template. These primers serve as a starting point for DNA synthesis. 

How does DNA polymerase synthesize DNA?

• Moves along the leading strand in the 5’ to 3’ direction

• In the lagging strand, in order to build it must move in the 3’ to 5’ direction

• Starts at the RNA primer

How is the lagging strand synthesized?

• In a discontinuous manner

• Primase synthesizes primers used by DNA polymerase for DNA synthesis (we need several primers for lagging strand, only one for the leading)

• These DNA fragments are called okazaki fragments

• The exonuclease enzymes removes RNA primers, DNA polymerase fills the gaps

Does the synthesis of the DNA strand occur at the same time?

• Yes, synthesis occurs at the same time, one occurs in the forward, direction, the other occurs in the reverse

What component of DNA replication contributes to telomere shortening and the DNA replication problem?

The short segment at the end of the DNA segment involves the initial placement of the RNA primer, however, it cannot be replaced with DNA by DNA polymerase since it can only synthesize DNA in the 5’ to 3’ direction.

• This contributes to telomere shortening

What are exogenous sources of DNA damage?

external sources

What are endogenous sources?

internal sources

What are some examples of endogenous damage that may affect DNA?

• Errors in replication

• Reactive oxygen species (A byproduct of metabolism)

What are some examples of exogenous damage that may affect DNA?

• Ultraviolet (UV 200 - 300 nm) radiation from the sun

• Other radiation (X-Rays and Gamma Rays)

• Mutagenic chemical (DNA intercalating agents)

How do healthy cells respond to DNA damage?

• Repairing the DNA using cells DNA repair mechanism

• Killing the cell via apoptosis

What happens to cells if DNA damage is not repaired?

DNA mutations arise

What are silent mutations?

• Have no affect on the protein as it does not change the amino acid sequence

What are mis-sense mutations?

• Result in the incorporation of the wrong amino acid

What are nonsense mutations?

• Mutations resulting in a premature stop codon, therefore results in a premature or truncated protein

What are frameshift mutations?

• Changes the' ‘frame’ of protein translation

Endogenous DNA damage can be a result of what four error types?

1.) Base Loss

2.) Mismatched Bases

3.) Deamination

4.) Oxidation by ROS

Describe a base loss error.

Endogenous DNA Damage

• Glycosyl bond linking DNA bases with deoxyribose is labile (Unstable / can break easily)

• May lead to spontaneous loss of the bases purines or pyrimidines

• Causes formation of apurinic / apyrimidinic site

• Therefore when the DNA is replicated it can cause base pairing deletions and breaks

Describe a mismatched base error.

Endogenous DNA Damage

• Mis-incorporation of a nucleotide during replication (e.g. incorperating T instead of A)

• If DNA is replicated will cause point mutations

Describe a deamination error.

Endogenous DNA Damage

• Amino groups of the nucleic acid are unstable, if they were to lose an amino groups it can change the base identity

• Can cause point mutations when DNA is replicated

Describe the effect oxidation by ROS has on DNA.

Endogenous DNA Damage

• Reactive oxygen species result from oxidative metabolism and ionizing radiation

• Produces hyperactive oxygen or radicals that can modify nucleotides

• Can cause point mutations when replicated, common one is G-T mutation

Describe a methylation / alkylation error.

Exogenous DNA Damage

• Many environmental chemicals can modify DNA bases, by addition of a methyl (methylation) or alkyl group (Alkylation)

• Can cause point mutation when replicated

Describe an inter-strand or cross-link error.

Exogenous DNA Damage

• By attaching to bases on both strands, alkylating agents like cancer drugs can cross-link both strands

• May cause blocks in replication, can also cause breaks

• Cross-links can be generated by UV and ionizing radiation

Describe UV Light errors.

Exogenous DNA Damage

• Ultraviolet light is absorbed by the nucleic acid bases and the resulting influx of energy can induce chemical changes

• Most frequent photoproducts are pyrimidine dimers T/C

• Also can induce inter-strand crosslinks

• Can cause mis-pairing during replication

Describe Ionising radiation errors.

Exogenous DNA Damage

• Causes single strand and double strand breaks

• Breakage of chomosomes could cause translocations, duplications, amplifications, inversions, deletionds

How is the G1 DNA damage checkpoint activated?

DNA Damage Response (DDR) senses DNA lesions and activates the G1 DNA checkpoint via activation of p53

What do the repair pathways in most cells look like? (5 steps)

1.) Recognition

• Detection of the damage

2.) Incision

• Cutting into the damaged DNA

3.) Excision

• Removal of the damaged part

4.) Polymerisation

• Gap filled by DNA polymerase

5.) Religation

• Joining gaps by DNA ligase