18.01.05 DNA replication Flashcards
Why is DNA replication termed semi-conservative?
Only one strand of the each resultant daughter molecule is newly synthesised.
dsDNA unwinds to act as a template and each daughter DNA molecule contains one DNA strand from parent molecule and one newly synthesised strand.
Where does DNA replication initiate?
At origins of replication, using the origns recognition complex.
Replication begins at sites with open chromatin configurations. Induced local changes to chromatin to allow replication to being.
CDC45 recruite DNApol a and d and allows intiation or replication and repliosome assembly.
When does DNA replication occur?
During the S phase of the cell cycle.
Actively transcribed euchromatin is replicated early in S phase.
Heterochromatic regions are replicated later (Domino model of replication).
Describe the function or the origin recognition complex.
Binds origin of replication during G1
Functions with CDC6 and CDT1 to load MCM2-7 helicase onto DNA.
What is the role of the topoisomerases?
Creates a ‘nick’ in a single DNA strand to release tension that holds the DNA in coils and allows the double helix to be unwound by helicases to form the Y-shaped replication fork.
Topoisomerase I - nicks single strand
Topoisomerase II - nicks both strands
What is the role of helixase?
Unwinds the ds DNA at the replication form Recruited by ORC, CDC6 and CDT to the position of the form in a process called ‘licensing’ which with other proteins forms the pre-initiation complex.
What is the role of primases?
RNA polymerase that do not require a free 3’ OH
Attach small complementary RNA sequences as a primer at the replication forl.
Provide a 3’ OH group needed as a substrate by DNA polymerase to start synthesis.
What is the role of DNA polymerases?
TO synthesis new DNA strands
adding deoxynucleoside monophosphate (dNMP) to the free 3’OH group of growing DNA strand.
Where does the energy come from to make the phosphodiester bond of the DNA backbone?
hydrolysis of dNTPs.
How is the function of DNA polymerase limited?
- Can only act in one direction 5’ - 3’
- Can only act on single stranded DNA only (from helicase)
- Required a free 3’OH (provided from primase)
How many different types of DNA polymerase are there?
> 20, grouped into four families.
Describe the family B polymerases
High-fidelity DNA polymerases that replicate nuclear DNA
Include a 3’-5’ exonuclease proff reading function which enable incorrectly incorporated bases to be removed and repaired.
Gives three examples of family B polymerases and their roles.
DNA polymerase α (alpha) – complex of polymerase and primase that initiatewhics DNA synthesis and Okazaki fragments
DNA polymerase δ (delta)– main polymerase that synthesises most of the lagging strand, and is involved in DNA repair
DNA polymerase ε (epsilon) – main polymerase that synthesises most of the leading strand, and is involved in DNA repair
What is the role of DNA polymerase y (gamma)?
Replicated mitochondrial DNA.
In family A - others in family A are involved in DNA repair and recombination.
What are the roles of DNA polymerases in families X and Y?
Work in DNA repair and only synthesise small stretches of DNA.
Useful for synthesising opposite breaks in DNA (translation synthesis) and contributing to sequence diversity of immunoglobulins.
Why is DNA replication termed semi-discontinuous?
Strands of the parental DNA duplex are antiparallele but both daughter strands have to be synthesised in 5’ - 3’ direction and DNApol requires a free 3’OH which leads to
1) leading strand - synthesised continuously
2) lagging strand - synthesised discontinuously in small steps making Okazaki fragments (100-1000nt) which are covalently joined together by DNA ligase.
Describe two targets in DNA replication for anti-cancer therapy.
1) DNA replication requires a free pool of nucleotides. Antimetabolites act to inhibit the synthesis of new nucleotides e.g. 5-FU which inhibits synthesis of T and therefore DNA replication.
2) Doxorubicin - inhibits the action of topoisomerase and stops DNA replication preventing further division and resulting in cell death.
What is the function of single-stranded binding proteins? Give an example.
Maintain stability of ssDNA at replication fork and protects the vulnerable ssDNA from enzymatic attack.
E.g. BRCA2 protects newly synthesised DNA from degradation by the nuclease MRE11 when replicated forks are stalled (e.g. no free nucletodies).
BRCA2 bind RAD51 and promotes its assembly on ssDNA which prevents fork reversal. BRCA2 is phosphorylated when no longer required for fork protection and RAD51 filaments then dissociate which promote progression to mitosis.
What is the role of DNA ligases?
CAtalyse phosphodiester bond formation between unattached by adjacent 3’OH and 5’PO4 groups bewteen Okazaki fragments and after RNA primers are removed.
What is the end-replication problem?
At the extreme template end there is no template ahead of the replicating region for primase to copy and make the RNA primer for the lagging strand.
What is the role of telomerase?
Specialised reverse transcriptions (RNA-dependent DNA polymerase) solves the end-replication problem
What are the subunits of telomerase?
TERT - protein subunit
TERC - RNA subunit - CUAACCCUAACG which is complementary to telomere TTAGGG repeats.
How does telomerase work?
Binds to overhanging 3’ end of parental lagging strand and the TERC provides the template to extend the telomere of the parental strand by one repeat subunit (several times) - lagging strand now has room for primase and can be extended = whole region in replicated.
What is a T-loop?
Following telomerase activity, the lagging strand still cannot be extended to the extreme 5’ end, leaving an overhanging 3’ single stranded, non-coding G-rich strand of ~200 nts long (telomere). This can fold back, invade the double-stranded region and form base-pairs with the complementary (C-rich) strand to form a telomeric loop (T-loop). This is thought to protect telomere DNA from cellular mechanisms that repair double-stranded DNA breaks.
