Medical and Molecular Genetics
Lecture 10 DNA Synthesis
1) Define the terms primer and template.
Primer – the enzyme that synthesizes DNA(DNA polymerase) can only add
nucleotides to an already existing strand of DNA or RNA that is based-paired
with the template, i.e. the primer.
Template – The DNA strand that is copied into a complementary strand of DNA.
2) Explain the differences and the similarities between leading and lagging strand
synthesis.
Leading Strand
Lagging Strand
Synthesis is 5’ to 3’
Synthesis is 5’ to 3’
Continuously synthesized Discontinuously synthesized
Requires an RNA primer
Requires an RNA primer
Primer extention – occurs Primer extension – DNA polymerase links a dNTP onto the
the same as the lagging
3’ end of the primer and initiates lagging strand synthesis,
strand but only once per
its extended for 1,000 nucleotides until it contacts the 5’ end
template.
resulting in Okazaki fragments.
Primer removal – also
Primer removal – the RNaseH degrades the RNA portion of
occurs only once to
an RNA/DNA duplex and leaves a phosphate group a the 5’
remove the initial primer
end of the adjoining DNA segment.
DNA polymerase
Gap filling – DNA polymerase uses Okazaki fragment
DNA ligase at the starting Covalent joining of DNA fragments – the 3’ is joined to the
primer.
5’ end by DNA ligase.
3) List the functions of the cis and trans-acting factors required for DNA
replication. Multiple origins of replication in eukaryotes are cis-acting factors that
create bidirectional replication forks. Trans-acting factors include: Origin binding
protein regognizes the origin, denatures the DNA and binds to helicase; Helicase
unwinds the DNA double strand; Single-strand DNA binding protein (SSB) prevents
the unwound DNA from renaturing; Primase synthesizes RNA primers; DNA
polymerase replicates the template; Processivity factors help load and anchor the
polymerase; Topoisomerase removes the positive supercoils that form as the fork is
unwound; RNaseH removes RNA portions from Okasaki fragments; Ligase seals the
nicks.
4) Describe how leading and lagging strand synthesis is coordinated. The two
polymerases pointing in opposite directions are held together by a processivity
platform and each is bound to a second processivity factor. When the lagging strand
finishes an Okasaki fragment it disassociates and is directed to bind to the next primer
by another processivity factor.
5) Define what is meant by semidiscontinuous and semiconservative replication.
Semiconservative replication means that each of the two new duplexes consist of one
of the original parental strands (conserved) and a newly synthesized daughter strand.
Semicontinuous replication means the leading strand is continuously replicated while
the lagging strand is discontinuously replicated.
6) State the function of telomerase and describe its probable mechanism.
Telomerase replicates chromosomal tips by extending the 3’ ends of a chromosome
by adding numerous repeats of a six base pair sequence, which is primed by primase
and extended by polymerase. The telomerase recognizes the tips of chromosomes
with the sequence [TTAGGG]n. Telomerase consists of an RNA sequence
complimentary to TTAGGG and a protein. The telomerase binds to a two base
portion of the sequence on the 3’ end of the DNA and adds TTAGGG before
translocating 6 bases and adding another sequence.
7) List some examples of drugs that inhibit replication and state their mechanism
of action. Substrate analogs (dNTPS) can be incorporated into DNA by polymerase
and end replication due to their 3’ hydroxyl group which cannot bind:
Azidothymidine (AZT) terminates replication by replacing the 3’ hydroxyl group.
Acyclovir in an effective anti-herpes virus drug because the herpes thymidine kinase
phosphorylates the drug and lets it incorporate into its DNA thus stopping replication.
Intercalating agents are generally aromatic compounds that wedge themselves into
DNA and prevent the replication complex from binding thus producing mutagenic
errors: Anthracycline glycosides and actinomycin D. DNA damaging agents like
Cisplatin, which creates a platinum complex that cross-links adjacent guanine
residues, causes chemical damage. Topoisomerase inhibitors: Nalidixic acid and
fluoroquinolones (bacterial), and campothectin (anti-cancer).