DNA Replication- Lecture 29

Question 1

DNA replication begins at ______ which are located on average _____.

Answer 1

Origins of replication (oris)

100,000 base pairs apart

Question 2

_____ is the term used to describe initiation of DNA replication from an origin.

Answer 2

Origin firing

Question 3

Describe the pathway of bi-directional replication.

Answer 3

DNA polymerase synthesizes DNA using each strand as a template starting at an ori and the “replication fork” representing newly synthesized DNA moves away from the ori in both directions simultaneously without releasing for hundreds of thousands of nucleotides

Question 4

What are the two parts of the newly replicated DNA?

Answer 4

the leading and lagging (Okazaki fragments) strands

Question 5

What are the DNA polymerases involved in DNA replication?

Answer 5

alpha- primer synthesis (DNA primase activity)
beta- base excision repair
gamma- mitochondiral DNA polymerase
delta- major lagging strand synthesis and MMR function
epsilon- major leading strand synthesis and NER functions
zeta- inaccurate replication at thyamine dimer
eta- adds As opposite thyamine dimers

Question 6

What are the four phases of the cell cycle?

Answer 6

Gap phase (G1)
S phase (S)
Gap phase (G2)
Mitosis (M)

Question 7

What is the first step of DNA replication?

Answer 7

formation of the pre-initiation complex- binding at an ori by the Origin Recognition Complex (ORC) which recruits additional proteins (topoisomerase and single stranded DNA binding protein) and the replication machinery

Question 8

Unlike synthesis by RNA polymerase, DNA synthesis proceeds only from a _______ that provides a free ____ on to which DNA polymerase adds the next nucleotide.

Answer 8

pre-existing “primer”

3’ hydroxyl

Question 9

Describe the pathway of initiation?

Answer 9

DNA primase subunit of DNA polymerase alpha) lays down a short RNA primer, which is then extended by DNA polymerase (once for the leading strand and many times for the lagging) –> RNA primers are eventually replaced with DNA and all DNA fragments are ligated together to forma continuous double-helix from one end of the chromosome to the other

Question 10

How are RNA primers removed from Okazaki fragments?

Answer 10

“old” RNA primer (located ahead of a new primer extension product) is displaced by DNA polymerase –> “flap structure with the RNA no longer annealed to the DNA template strand –> RNA primer is cleaved off endonucleolytically by FEN1 (a flap endonuclease) –> nick between the 5’ end of the “old” Okazaki DNA and the 3’ end of the “new” Okazaki DNA is sealed by DNA ligase

Question 11

_____ are commonly used as antivirals.

Answer 11

Nucleoside analogues that do not have a 3’ hydroxyl onto which a subsequent nucleotide can be added

Question 12

The ends of chromosomes are called _____.

Answer 12

telomeres

Question 13

What is the repeat sequence for telomeric DNA?

Answer 13

TTAGGG

Question 14

How is the telomeric region distinguished from a broken DNA end?

Answer 14

the folding of the DNA back into a structure called a t-loop

Question 15

Why are t-loops important?

Answer 15

prevents end-to-end joining of chromosomes and signal that an end is not broken (which indicates catastrophic DNA damage that can result in apoptosis)

Question 16

How are telomere repeat sequences added?

Answer 16

by telomere-specific proteins that form the “shelterin” complex

Question 17

What is the end replication problem?

Answer 17

the nature of the lagging strand does not allow for the very end to be fully synthesized, resulting in the telomere shortening with each round of replication

Question 18

How do cells solved the end replication problem?

Answer 18

a reverse transcriptase known as “telomerase” (a ribonucleoprotein complex that carries its own RNA template and that can extend the lagging strand)

Question 19

Why do only some cells have telomerase activity?

Answer 19

because normal somatic cells do not need to continually replicate, so they are limited to a number of cell divisions before they become senscent (the Hayflick limit)

Question 20

In some cancers, the reactivation of _____ allows cells to continue to divide unchecked.

Answer 20

telomerase

Question 21

What is the mechanism of telomerase based DNA extension?

Answer 21

telomerase RNA binds to a complementary sequence in the lagging strand template –> lagging strand template is extended by six nucleotides based on the sequence of the RNA template carried by telomerase –> telomerase binds again at its complementary sequence (six nucleotides downstream of the last one) and the reaction is repeated