18.01.05 DNA replication

Question 1

Why is DNA replication termed semi-conservative?

Answer 1

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.

Question 2

Where does DNA replication initiate?

Answer 2

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.

Question 3

When does DNA replication occur?

Answer 3

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).

Question 4

Describe the function or the origin recognition complex.

Answer 4

Binds origin of replication during G1

Functions with CDC6 and CDT1 to load MCM2-7 helicase onto DNA.

Question 5

What is the role of the topoisomerases?

Answer 5

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

Question 6

What is the role of helixase?

Answer 6

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.

Question 7

What is the role of primases?

Answer 7

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.

Question 8

What is the role of DNA polymerases?

Answer 8

TO synthesis new DNA strands

adding deoxynucleoside monophosphate (dNMP) to the free 3’OH group of growing DNA strand.

Question 9

Where does the energy come from to make the phosphodiester bond of the DNA backbone?

Answer 9

hydrolysis of dNTPs.

Question 10

How is the function of DNA polymerase limited?

Answer 10

1. Can only act in one direction 5’ - 3’
2. Can only act on single stranded DNA only (from helicase)
3. Required a free 3’OH (provided from primase)

Question 11

How many different types of DNA polymerase are there?

Answer 11

> 20, grouped into four families.

Question 12

Describe the family B polymerases

Answer 12

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.

Question 13

Gives three examples of family B polymerases and their roles.

Answer 13

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

Question 14

What is the role of DNA polymerase y (gamma)?

Answer 14

Replicated mitochondrial DNA.

In family A - others in family A are involved in DNA repair and recombination.

Question 15

What are the roles of DNA polymerases in families X and Y?

Answer 15

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.

Question 16

Why is DNA replication termed semi-discontinuous?

Answer 16

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.

Question 17

Describe two targets in DNA replication for anti-cancer therapy.

Answer 17

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.

Question 18

What is the function of single-stranded binding proteins? Give an example.

Answer 18

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.

Question 19

What is the role of DNA ligases?

Answer 19

CAtalyse phosphodiester bond formation between unattached by adjacent 3’OH and 5’PO4 groups bewteen Okazaki fragments and after RNA primers are removed.

Question 20

What is the end-replication problem?

Answer 20

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.

Question 21

What is the role of telomerase?

Answer 21

Specialised reverse transcriptions (RNA-dependent DNA polymerase) solves the end-replication problem

Question 22

What are the subunits of telomerase?

Answer 22

TERT - protein subunit

TERC - RNA subunit - CUAACCCUAACG which is complementary to telomere TTAGGG repeats.

Question 23

How does telomerase work?

Answer 23

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.

Question 24

What is a T-loop?

Answer 24

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.

Question 25

Give examples of three disease related to issues with DNA replication.

Answer 25

1) Meier Gorlin syndrome - Origin of replication defect
2) Bloom syndrome - helicase defect
3) Hutchinson-Gilford progeria syndrome (HGPS) - polymerase defect
4) Mandibular hypoplasia, deafness, progeroid features and lipodystrophy (MDPL) syndrome - polymerase defect
5) Dyskeratosis congenita (DKC) - telomerase defect

Question 26

What are the clinical features and causes of Merir-Gorlin syndrome?

Answer 26

Defect in the origin of replication
AR primordial dwarfism syndrome
Caused by mutations in ORC subunits; Orc1, Orc4, Orc6, Cdt1, Cdc6
Defect in G1 with an inability to procced to S phases causes reduced growth rate.
Lack or ORC1 protein leads to inability to initiate replication origins and decreases pre=replication complex assembly

Question 27

What are the clinical features and causes of Hutchinson-Gilford progeria syndrome (HGPS)

Answer 27

Defect in polymerase.

Premature ageing disorder, hallmark phenotype is early replication arrest.

Replication factor C (RFC) binds the 3’ end of the primed DNA strand and clamps Proliferating Cell Nuclear Antigen (PCNA) to DNA, which holds polymerase δ to DNA.

RFC1 (the large subunit of RFC) contains the major DNA binding domains of RFC and interacts directly with PCNA.

In HGPS cells RFC1 is cleaved. Truncated RFC1 is defective in loading PCNA and polymerase onto DNA for replication – assembly of the replication machinery is disrupted and the replication fork stalls and collapses, leading to early replication arrest.

HGPS is caused by a point mutation in the LMNA gene, resulting in production of farnesylated Lamin A/truncated Lamin A (aka Progerin) which is somehow involved in RFC1 cleavage.

In HGPS cells exposed to serine protease inhibitor RFC1 is not cleaved ?possible treatment target

Question 28

What are the clinical features and causes of Mandibular hypoplasia, deafness, progeroid features and lipodystrophy (MDPL) syndrome)?

Answer 28

Polymerase defect

Germline mutations in POLD1 and POLE cause polymerase proofreading-associated polyposis (PPAP) and MDPL Syndrome.

Somatic mutations in POLD1 and POLE have also been found in sporadic colorectal and endometrial cancer (Ref: Heitzer E and Tomlinson I Curr Opin Genetics and Development 2014 24:107-113)

Question 29

What are the clinical features and causes of 
Dyskeratosis congenita (DKC)?

Answer 29

Telomerase defect

Rare progressive congenital disorder that in some ways resembles premature aging.

8 DKC genes (DKC1, TERC, TERT, NOP10, NHP2, TIN2, C16orf57, and TCAB1) having been characterized.

Sufferers of DKC have been shown to have a reduction in TERC levels invariably affecting the normal function of telomerase - telomere maintenance during development suffers

exogenous TERC alone can correct the telomerase defect, restore telomere length, and improve cell growth in DKC and TERC-mutated lymphocytes. Possible future treatment for humans