1-6 DNA Replication

Question 1

Replication occurs in the _____ direction of the ____ strand. How does it move compared to the fork?

Answer 1

5’—>3’ direction of the leading strand, same direction

Question 2

the strand which is extended continuously

Answer 2

the leading strand

Question 3

new nucleotides are added to the ____ end during replication

Answer 3

3’ end

Question 4

the strand which is replicated discontinuously, forming ___

Answer 4

lagging strand, okazaki fragments

Question 5

Initiation takes place at the ____, which is marked by a protein complex called _______ which is responsible for?

Answer 5

origin of replication, origin replication complex (ORC), recruiting other initiation proteins to the origin

Question 6

What does DNA helicase do?

Answer 6

aka MCM, unwinds the double stranded DNA

Question 7

what prevents the exposed strands of the unwound dna from reannealing?

Answer 7

the binding of single-stranded dna (ssdna)binding protein (RPA)

Question 8

what does primase do?

Answer 8

it is a specialized RNA polymerase that lays down an RNA primer because DNA polymerase can’t work without it

Question 9

how many primers are necessary for the leading strand? lagging strand?

Answer 9

leading strand - continuous, just one

lagging strand - discontinuous, multiple

Question 10

what does POL alpha do?

Answer 10

makes a complex with primase, takes over the RNA primer and synethesizes a short stretch of DNA which then is taken over by DNA polymerase to synthesize longer stretches of DNA

Question 11

which DNA polymerase is for what strand?

Answer 11

DNA polymerase ε for leading strand. (E for leading)

DNA polymerase δ for lagging strand

Question 12

what is PCNA?

Answer 12

a sliding clamp, loaded by clamp loader RC-F that keeps dna polymerase firmly on the DNA template

Question 13

RC-F?

Answer 13

loads the clamp(PCNA) that keeps dna polymerase tightly engaged to dna template

Question 14

How is the lagging strand turned to a continuous piece of DNA?

Answer 14

the RNA primer and some DNA polymerase alpha synthesized DNA are removed:

1. DNA polymerase δ genetrates a flap by displacing the DNA pol-alpha synthesized DNA and primer.
2. Flap cut by flap endonuclease (FEN-1)
3. template gap is filled by DNA polymerase δ
4. gap between adjacent nascent strands sealed through phosphodiester bonds via DNA LIGASE

Question 15

Define the mechanism limiting chromosome DNA replication to once per cell cycle

Answer 15

When S phase begins, Orc1 (origin recognition complex) is suppressed or degraded through phosphorylation by cyclin A/Cdk2. Cdt1 is also degraded, and any that remains is sequestered by geminin. Finally, Cdc6 is phosphorylated by cyclin A/Cdk2 and removed from the nucleus.

Question 16

Describe the nucleic acid and protein components of chromosomal DNA replication and their coordinated composition at the replication fork.

Answer 16

Replication forks begin at origins of replication. Replication itself begins with a pre-replicative complex (pre-RC). Orc1 (origin recognition complex) recognizes the origin and recruits other replisome proteins to form the pre-RC, including the MCM2-7 complex (which includes helicase), Cdt1, and Cdc6. Helicase unwinds the double helix structure. The DNA primase/DNA polymerase alpha complex allows for the placement of an RNA primer and the synthesis of new DNA along the original copy. ssDNA binding protein stabilizes the unwound ssDNA. DNA polymerase epsilon is the polymerase for the leading strand, and DNA polymerase delta is the polymerase for the lagging strand. PCNA (the clamp protein) engages epsilon and delta onto the strands. RF-C (the clamp loader) allows for new clamp protein assembly. Ligase ligates the Okazaki fragments together.

Question 17

explain different mechanisms of replication stress-induced genome instability

Answer 17

In cancer cells, expression of pre-RC proteins is increased

Question 18

explain different mechanisms of replication stress-induced genome instability

Answer 18

1. In cancer cells, expression of pre-RC proteins is increased
2. if the cell runs out of dNTP’s (deoxynucleotides) during replication, fork needs to be protected until dNTP supply is restored and replication can be reinitated
3. intereference between transcription and translation occurs more frequently on longer genes and leads to stress.
4. If a cell cycle checkpoint is reached, and DNA damage not repaired, the DNA damage response (DDR) pathway is initiated. ATM protein kinase is recruited to activate p53, which leads the cell to senescence or apoptosis.

Question 19

unprotected replication forks give rise to..

Answer 19

formation of single stranded DNA gaps and ultimately doubel strand breaks.

Question 20

what are CFS?

Answer 20

chromosomal fragile sites - genomic loci that are hot spots for genomic rearrangements and are implicated in diseases ranging from cancer to neurological disorders.

Question 21

replication of DNA requires a primer for DNA synthesis because

Answer 21

DNA polymerase requires a base-paired nucleotide with a free 3’ end before it can add a new nucleotide

Question 22

molecule of bacterial DNA introduced into yeast cell is imported into nucleus but fails to replicate. Which yeast protein is likely unable to use the bacterial DNA as a substrate?

Answer 22

Initiator Proteins

Question 23

Takes 40 minutes for e.coli chromosome to completely replicate. How are bacterial cells able to divide every 20 minutes? How can cells divide more rapidly, apparently, than their DNA can be copied?

Answer 23

e.coli origin of replicaion initiates a second round of replication before the first is complete

Question 24

mutations in pre-RC components as well as over-expression of pre-RC components can both lead to ______. How?

Answer 24

genome instability

mutations in pre-RC components reduce/inhibit origin activation whereas over-expression of pre-RC components can lead to re-replication.

Question 25

Describe the biological importance of replication timing control.

Answer 25

Genes that are transcribed more frequently will be replicated earlier on. This regulation is important for development of certain cells. For example, as pro-B cells develop into mature B cells, they need to rearrange certain loci, including the IgH locus, in order to generate particular immune receptors. Early replication of the IgH locus is necessary to achieve this rearrangement. Asynchronous gene replication is implicated in X-inactivation and genomic imprinting (expressing certain copies of genes and not their homologous copies): two cases in which one copy of a length of genetic material is replicated before its homologous copy.

Question 26

replication fidelity is due mostly to?

Answer 26

the proofreading activity of DNA polymerase

Question 27

describe the polymerase switch event

Answer 27

primase in complex with pol-a, which takes over RNA primer and lays down a short stretch of DNA.

Pol alpha is then replaced by a more processive (able to be engaged on DNA template for longer periods) dna polymerase (sigma for leading, & for lagging)

Question 28

enzymes involved in telomere replication

Answer 28

TERT and TERC.

TERC is an RNA template that TERT can use to add multiple copies of DNA at the 3’ template of the lagging strand. lagging strand then gets completed by dna polmerase

Question 29

In BER, lesion is removed by

Answer 29

dna glycosylase

Question 30

aicardi goutieres syndrome

Answer 30

mutation in the RNASEH2A gene