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Path 19.01 and 19.07 > 19.01.05 DNA replication > Flashcards

19.01.05 DNA replication Flashcards

DNA replication

1
Q

three phases of DNA replication

A

Initiation, Elongation and Termination.

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2
Q

DNA replication is

A

semi-conservative

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3
Q

What happens during DNA replication

A

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.

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4
Q

DNA Replication initiates at a specific spot called

A

origins of replication

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5
Q

Direction of replication

A

5’ to 3’. Need a free hydroxyl group (OH-) to start synthesising

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6
Q

Origin recognition complex (ORC) – Binds origin of replication during which cell cycle phase

A

G1

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7
Q

ORC acts with CDC6 and CDT1 to load what onto DNA

A

MCM2-7 helicase. This is the pre-replication complex

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8
Q

Pre-replication complex binds with CDC45 and GINS complex to form what

A

the pre-initiation complex.

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9
Q

The preinitiation complex requires cyclin-dependent kinase activity, so can be inhibited by

A

CDK-inhibitors

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10
Q

CDC45 recruits what

A

DNA polymerases alpha and delta

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11
Q

What do DNA polymerases alpha and delta do?

A

allow initiation of replication and assembly of repliosome

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12
Q

what is the repliosome

A

complex of polymerase and associated proteins at the replication fork

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13
Q

what do topoisomerases do

A

create a nick in a single DNA strand.

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14
Q

What do helicases do

A

unwind DNA at replication fork

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15
Q

WHat shape is replication fork

A

Y

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16
Q

Difference between topoisomerase 1 and 2

A

1 nicks a single strand, 2 nicks both strands

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17
Q

Doxorubicin is an anticancer chemotherapeutic that acts by

A

inhibiting topoisomerase 2 enzymes and thus stop DNA replication, leading to cell death.

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18
Q

ORC, CDC6 and CDT recruit what to the fork in a process known as licensing

A

helicase. Complex is known as pre-initiation complex.

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19
Q

what is a primase

A

DNA polymerase that doesn’t require a free 3’ OH group

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20
Q

what do primase do

A

attach small complementary RNA sequences as a primer at the replication fork

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21
Q

What do the RNA primers do

A

provide a 3’ OH group needed as a substrate by DNA polymerases to start synthesis

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22
Q

DNA polymerases do what

A

synthesise new DNA strands

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23
Q

how do polymerases work

A

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

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24
Q

what are dNTPs

A

deoxynucleoside triphosphates. Used as substrates for DNA polymerases. Energy stored as triphosphate is used to make the phosphodiester bond

25
Q

3 limiting factors for DNA polymerase function

A

One direction (5’ to 3’), acts on signle stranded DNA only (provided by helicase), needs a 3’ end (provided by primase)

26
Q

How many type of DNA polymerases

A

20.

27
Q

How groups are DNA polymerases are they grouped into

A

4 (A, B, X, Y)

28
Q

Family A DNA polymerase group includes

A

DNA pol gamma, replicates mitochondrial DNA

29
Q

Family B DNA polymerase group includes

A

high-fidelity, polymerases that replicate nuclear DNA. e.g. DNA polymerase alpha, delta and epsilon

30
Q

What is 3’-5’ exonuclease proof reading function

A

to enable incorrectly incorporated bases to be removed and repaired

31
Q

DNA polymerase alpha

A

initiates DNA synthesis and Okazaki fragments

32
Q

DNA polymerase delta

A

main polymerase that synthesises most of lagging strand

33
Q

DNA polymerase epsilon

A

main polymerase that synthesises most of leading strand

34
Q

DNA polymerase delta and epsilon also involved in

A

DNA repair. e.g. Nucleotide excision repair, base excision repair, mismatch repair, double strand break repair, break induced recombination.

35
Q

DNA polymerase delta and epsilon composed of

A

heterotetramers, containing catalytic and accessory subunits

36
Q

changes in expression/ activity in POLD1 linked to

A

senescence and aging. Somatic changes of POLD1 and POLE linked to sporadic colorectal and endometrial cancer

37
Q

X and Y families of polymerases often have

A

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

38
Q

Why is replication semi-discontinuous

A

Parental DNA strands are antiparallel, but both daughter strands have to be synthesised in the same direction- 5’ to 3’

39
Q

Leading strand is synthesised

A

continuously

40
Q

Lagging strand is synthesised

A

discontinuously

41
Q

What is an Okasaki fragment

A

Segments of DNA 100-1000 nucleotides

42
Q

How are Okasaki fragments joined

A

covalently joined by DNA ligase.

43
Q

Anticancer drugs called antimetabolites inhibit synthesis, how?

A

Inhibit synthesis of nucleotides, therefore inhibiting DNA replication. 5-FU drug inhibits synthesis of thymidine nucleotides

44
Q

what is the function of single-stranded binding proteins

A

Maintain stability of single stranded DNA, protects from nucleases

45
Q

Example of a single-stranded binding protein

A

BRCA2. Protects newly synthesised DNA from degradation when replication forks are stalled (eg if insufficient pool of nucleotides).

46
Q

What do DNA ligases do

A

Catalyse phosphodiester bond formation between adjacent 3’OH and 5’ phosphate groups.

47
Q

end replication problem is

A

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

48
Q

What is telomerase

A

Reverse transcriptase enzyme. RNA-dependent DNA polymerase.

49
Q

Telomerase TERT subunit is

A

telomerase reverse transcriptase (TERT) – protein subunit

50
Q

Telomerase TERC subunit is

A

telomerase RNA component (TERC) – RNA subunit. Complementary to telomere TTAGGG repeats.. It is a tandem repeat sequence (CUAACCCUAACG)

51
Q

How does telomerase function

A

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.

52
Q

Can lagging strand be extended to extreme 5’ end

A

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).

53
Q

Function of telomeric loop (T-Loop)

A

to protect telomere DNA from cellular mechanisms that repair double stranded DNA.

54
Q

Is telomerase active in adult somatic cells

A

No, so telomeres gradually shorten. Linked to aging. Cancer cells can activate telomerase to lead to uncontrolled replication.

55
Q

Meier-Gorlin syndrome caused by what genetic defect

A

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

56
Q

Bloom syndrome

A

Helicase defect (BLM protein has helicase activity). Causes chromosome instabilities due to defective DNA replication. Also increased SCE rate. Patients have growth retardation.

57
Q

Hutchinson-Gilford progeria syndrome

A

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.

58
Q

Dyskeratosis congenita (DKC)

A

Resembles premature aging. 8 genes (DKC1, TERC, TERT etc). Patients have reduced TERC levels.

Path 19.01 and 19.07 (45 decks)

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