19.01.05 DNA replication Flashcards
DNA replication
three phases of DNA replication
Initiation, Elongation and Termination.
DNA replication is
semi-conservative
What happens during DNA replication
double strand parent DNA unwinds to act as a template and each daughter duplex contains one DNA strand from parent molecule and one newly synthesised strand. 2 daughter DNA duplexes are identical to the parent duplex.
DNA Replication initiates at a specific spot called
origins of replication
Direction of replication
5’ to 3’. Need a free hydroxyl group (OH-) to start synthesising
Origin recognition complex (ORC) – Binds origin of replication during which cell cycle phase
G1
ORC acts with CDC6 and CDT1 to load what onto DNA
MCM2-7 helicase. This is the pre-replication complex
Pre-replication complex binds with CDC45 and GINS complex to form what
the pre-initiation complex.
The preinitiation complex requires cyclin-dependent kinase activity, so can be inhibited by
CDK-inhibitors
CDC45 recruits what
DNA polymerases alpha and delta
What do DNA polymerases alpha and delta do?
allow initiation of replication and assembly of repliosome
what is the repliosome
complex of polymerase and associated proteins at the replication fork
what do topoisomerases do
create a nick in a single DNA strand.
What do helicases do
unwind DNA at replication fork
WHat shape is replication fork
Y
Difference between topoisomerase 1 and 2
1 nicks a single strand, 2 nicks both strands
Doxorubicin is an anticancer chemotherapeutic that acts by
inhibiting topoisomerase 2 enzymes and thus stop DNA replication, leading to cell death.
ORC, CDC6 and CDT recruit what to the fork in a process known as licensing
helicase. Complex is known as pre-initiation complex.
what is a primase
DNA polymerase that doesn’t require a free 3’ OH group
what do primase do
attach small complementary RNA sequences as a primer at the replication fork
What do the RNA primers do
provide a 3’ OH group needed as a substrate by DNA polymerases to start synthesis
DNA polymerases do what
synthesise new DNA strands
how do polymerases work
Add deoxynucleoside monophosphate (dNMP) to free 3’ OH group of growing DNA strand.
what are dNTPs
deoxynucleoside triphosphates. Used as substrates for DNA polymerases. Energy stored as triphosphate is used to make the phosphodiester bond
3 limiting factors for DNA polymerase function
One direction (5’ to 3’), acts on signle stranded DNA only (provided by helicase), needs a 3’ end (provided by primase)
How many type of DNA polymerases
20.
How groups are DNA polymerases are they grouped into
4 (A, B, X, Y)
Family A DNA polymerase group includes
DNA pol gamma, replicates mitochondrial DNA
Family B DNA polymerase group includes
high-fidelity, polymerases that replicate nuclear DNA. e.g. DNA polymerase alpha, delta and epsilon
What is 3’-5’ exonuclease proof reading function
to enable incorrectly incorporated bases to be removed and repaired
DNA polymerase alpha
initiates DNA synthesis and Okazaki fragments
DNA polymerase delta
main polymerase that synthesises most of lagging strand
DNA polymerase epsilon
main polymerase that synthesises most of leading strand
DNA polymerase delta and epsilon also involved in
DNA repair. e.g. Nucleotide excision repair, base excision repair, mismatch repair, double strand break repair, break induced recombination.
DNA polymerase delta and epsilon composed of
heterotetramers, containing catalytic and accessory subunits
changes in expression/ activity in POLD1 linked to
senescence and aging. Somatic changes of POLD1 and POLE linked to sporadic colorectal and endometrial cancer
X and Y families of polymerases often have
high error rates. Tolerated as they function in DNA repeir process so only synthesise small stretches of DNA. Contribute to the sequence diversity of immunoglobulins
Why is replication semi-discontinuous
Parental DNA strands are antiparallel, but both daughter strands have to be synthesised in the same direction- 5’ to 3’
Leading strand is synthesised
continuously
Lagging strand is synthesised
discontinuously
What is an Okasaki fragment
Segments of DNA 100-1000 nucleotides
How are Okasaki fragments joined
covalently joined by DNA ligase.
Anticancer drugs called antimetabolites inhibit synthesis, how?
Inhibit synthesis of nucleotides, therefore inhibiting DNA replication. 5-FU drug inhibits synthesis of thymidine nucleotides
what is the function of single-stranded binding proteins
Maintain stability of single stranded DNA, protects from nucleases
Example of a single-stranded binding protein
BRCA2. Protects newly synthesised DNA from degradation when replication forks are stalled (eg if insufficient pool of nucleotides).
What do DNA ligases do
Catalyse phosphodiester bond formation between adjacent 3’OH and 5’ phosphate groups.
end replication problem is
when at the extreme end of the linear DNA molecule (end of a chromosome) there isn’t a template ahead of the replication region. So primase cannot copy to make an RNA primer for lagging strand
What is telomerase
Reverse transcriptase enzyme. RNA-dependent DNA polymerase.
Telomerase TERT subunit is
telomerase reverse transcriptase (TERT) – protein subunit
Telomerase TERC subunit is
telomerase RNA component (TERC) – RNA subunit. Complementary to telomere TTAGGG repeats.. It is a tandem repeat sequence (CUAACCCUAACG)
How does telomerase function
Telomerase binds to the overhanging 3’ end of parental lagging strand. TERC provides template to extend the telomere of parental strand by one repeat subunit (repeats several times). Gives room for primase to extend.
Can lagging strand be extended to extreme 5’ end
No. There will be a 3’ single strand overhang, ~200 nucleotides (non-coding G-rich telomere). Folds back and base pairs with the complementary (C-rich) strand to form a telomeric loop (T-Loop).
Function of telomeric loop (T-Loop)
to protect telomere DNA from cellular mechanisms that repair double stranded DNA.
Is telomerase active in adult somatic cells
No, so telomeres gradually shorten. Linked to aging. Cancer cells can activate telomerase to lead to uncontrolled replication.
Meier-Gorlin syndrome caused by what genetic defect
Origin of replication defect. Mutations in ORC1, 4, 6, CDT1, CDC6. Abberations leading to slower cell growth. Cells have a G1 defect so cannot proceed to S-phase. Autosomal recessive
Bloom syndrome
Helicase defect (BLM protein has helicase activity). Causes chromosome instabilities due to defective DNA replication. Also increased SCE rate. Patients have growth retardation.
Hutchinson-Gilford progeria syndrome
Premature aging disorder due to mutations in replication factor C (RFC), which binds to 3’ end of primerd DNA and sticks PCNA (proliferating cell cnulear antigen) to DNA. This holds polymerase delta to DNA. Truncated RFC1 leads to defective PCNA loading and polymerase onto DNA, leading to early replication arrest. Point mutations in LMNA lead to RFC1 cleavage.
Dyskeratosis congenita (DKC)
Resembles premature aging. 8 genes (DKC1, TERC, TERT etc). Patients have reduced TERC levels.
