Replication

Question 1

Semi-conservative and conservative

Answer 1

meselson and stahl experiment showed semi conservative replication where an original strand is present in each copy

Question 2

DNA polymerase 1. Bacteria

Answer 2

5’ to 3’. Replication and repair. Major activity. polA. Removes the primer (exonuclease activity)

Question 3

DNA Polymerase II. Bacteria

Answer 3

5’ to 3’. Repair. Not involved in replication

Question 4

DNA Polymerase III. Bacteria

Answer 4

5’ to 3’. Replication. Complex structure. Key enzyme. dnaE (polC). Synthesises from the primer.

Question 5

Okazaki fragments

Answer 5

DNA replication only in 5’ to 3’ direction. Leading strand and lagging strand where the fragments are

Question 6

Primer

Answer 6

15-30 bases. Polymerases need to start replication.

Question 7

Primosome

Answer 7

protein complex responsible for creating RNA primers on single stranded DNA during DNA replication

Question 8

Primosome structure

Answer 8

DNAB – helicase, unwinds DNA
SSB – single stranded binding protein protects single stranded DNA
DNAG – synthesises primer
Gyrase – releases stress in DNA by unwinding and rewinding

Question 9

Polymerase III structure

Answer 9

Left hand side does leading strand, right handed side does okazaki fragment.
Beta – clamps to DNA
Alpha – Synthesises DNA
Tau – holds units together
E – 5’ -3’ exonuclease
Green subunit on right reload enzyme back on to DNA

Question 10

SV40 replication. T antigen

Answer 10

Hexamer, acts as helicase and loader

Question 11

SV40 replication. RPA

Answer 11

Heterotrimic protein, binds to single stranded DNA and protects

Question 12

SV40 replication. Pol delta, PCNA, Rfc, Pol epsilon

Answer 12

Synthesise the leading strand. PCNA (poliferating cell nuclear antigen) is clamp, Replication factor c (RFC) 5 subunit protein that acts as clamp loader, catalyses the loading of PCNA onto DNA. Uses ATP and clamps to 3’ end, Pol delta is catalysis (same as dnapolymerase III core) as is Pol epsilon

Question 13

SV40 replication. Pol alpha, primase

Answer 13

Synthesise lagging strand. Then joined by Pol delta, PCNA, Rfc which extends the primer and syntheises most of the Okazaki fragments.

Question 14

Large chromosome replication

Answer 14

must be coordinated, many origins and each must only fire once per cell cycle, Must have a control system, synchronised to the cell cycle. Must be flexible to allow different patterns of replication in different cell types.
Concept of ‘licensing’ replication origins developed

Question 15

Telomer shortening

Answer 15

Synthesis of leading strand can continue all the way to the end. However the lagging strand cannot continue to the end because it requires an upstream primer. This means that the RNA primer is left on the lagging strand. This means that the daughter strand will always be shortened. Countered by a telomerase

Question 16

Telomerase action

Answer 16

Single strand at end (5’ to 3’). Telomerase associated RNA template binds to strand and catalyses reverse transcription in 5’ to 3’ direction - elongating telomere. The DNA strand is thought to slip and so more RNA template is available. The lagging DNA is therfore elongated more. This can be translocated and hybridised again and again. Primase then added and okazaki fragments can start from this lagging strand to prevent shortening

Question 17

Licensing factor

Answer 17

In the nucleus it allows replication. Degraded after replication. Licenses the origins of replication

Question 18

See lecture for control of replication

Answer 18

see it