3.) DNA Replication and Repair

Question 0

What is a replication fork?

Answer 0

.

Question 1

What is semiconservative replication?

Answer 1

Means that the products of DNA replication results in a hybrid strand, one new strand (daughter strand) and one of the original strands (parent strand).

Question 2

What is an origin of replication?

Answer 2

.

Question 3

What is the leading strand in DNA replication?

Answer 3

.

Question 4

What is the lagging strand in DNA replication?

Answer 4

.

Question 5

What is an Okazaki fragment?

Answer 5

.

Question 6

DNA synthesis always occurs in the ________ direction, and therefore reading of the strand occurs in the _______ direction.

Answer 6

5’ —> 3’

3’ —> 5’

Question 7

DNA polymerase requires ____________ (multiple things) for DNA synthesis.

Answer 7

1. ) Template strand

2. ) A primer

Question 8

What important substrates are needed for DNA synthesis?

Answer 8

1. ) Template strand that has a dNMP with a free 3’OH

2. ) dNTP that is complementary to the template strand.

Question 9

What is the main DNA polymerase for DNA synthesis?

Answer 9

DNA polymerase III

Question 10

During DNA synthesis, describe the general mechanism for the addition of a nucleotide base pair.

Answer 10

The 3’ OH of a dNMP on the DNA strand will act as a nucleophile and attack the alpha phosphate on the 5’ carbon of a dNTP, releasing pyrophosphate.

Question 11

What mechanisms contribute to the fidelity of DNA replication.

Answer 11

1. ) Nucleotide base pairing

2. ) DNA polymerase I exonuclease activity

Question 12

What is proofreading in DNA synthesis? Describe how it works. Is there a difference between Prokaryotic and Eukaryotic proofreading?

Answer 12

Proofreading is the correction of mismatched base pairs that occur during DNA synthesis. DNA Polymerase III has a 3’—>5’ exonuclease site in addition to its polymerase site. A mismatched base pair will block further polymerization. Polymerase will then slide back and position the nucleotide into the exonuclease active site. The nucleotide is removed and then DNA polymerase can continue with polymerization.

Question 13

What are the three general steps of DNA synthesis?

Answer 13

1. ) Initiation
2. ) Elongation
3. ) Termination

Question 14

The E. Coli site of initiation is the…

Answer 14

OriC sequence.

Question 15

Describe the process of initiation.

Answer 15

A DNA protein (DnaA) will recognize the site of initiation (aka the origin sequence aka DUE sequences) and will bind to this site. Using ATP the DnaA protein will induce tortional stress into the DNA strand, resulting in the opening of the DNA strand. This creates 2 binding sites for DNA helicase, which will then further unwind the strand. The binding of DNA helicase is mediated by another ATP dependent protein, which uses an ATP to bind the helicase to the DNA strand.

Note that the process of opening the strand relieves the tension that was introduced by DnaA protein. However, as further denaturation occurs, more tension will be introduced and will require a topoisomerase to relieve this tension.

Question 16

True or False: DNA initiation sequences are highly variable among different species.

Answer 16

False. They are highly conserved.

Question 17

The DUE sequences are rich in _______. What is the implication here?

Answer 17

DUE = DNA unwinding element.

Rich in AT’s, which are less stable than GC’s. This means that these sites will open easily

Question 18

DUE sequences cannot be __________. Meaning that they will convey direction.

Answer 18

palindromic.

Question 19

Describe the process of elongation.

Answer 19

DNA helicase is bound, and on the connected double strand a DNA topoisomerase binds to help relieve the tension that is introduced by unwinding the DNA. At this point we have a leading strand and lagging strand.
Leading strand can be used continuously as a template, requiring only DNA polymerase activity after a primer is placed down by DNA primase.

The lagging strand require the formation of Okazaki fragments. DNA primase will lay down, incrementally, a series of primers that will be the substrate for DNA polymerases. DNA replication will then occur in the opposite direction of the movement of the replication fork, and it will dissociate once it reaches the next primer. A SSB (single stranded binding protein) will prevent aggregation of the strands. DNA ligase will then connect these strands at the “nicks”.

There is more needed here based on his slides 35-37, also need to note orientation of the strand in the DNA polymerase complex.

Question 20

Describe the process of termination.

Answer 20

The DNA genome has “ter sequences” which are sequences to indicate termination. They are bound by tus (terminus utilization substance) proteins. Once the replication forks reach these sequences, they promote dissociation leaving catenated chromosomes. these are highly tangled structures, which require topoisomerase to relieve the tension and untangle the two separate DNA strands to yield two DNA’s.

The sequences occur in clusters and have opposite orientation. Bacteria have circular DNA, so the two replication forks will eventually reach each their own respective “fork traps.”

Question 21

The difference between Prokaryotic and Eukaryotic DNA replication is…

Answer 21

Eukaryotes will have multiple sites of initiation.

Question 22

Eukaryotes have what structure at the site of origin for DNA replication?

Answer 22

ORC: Origin replication Complex.

Question 23

How is DNA replication synchronized with the cell cycle? What are the points of regulation?

Answer 23

ORC is bound to the site of origin, and promotes binding of the DNA helicase complex known as the minichromosome maintenance protein (MCM, analagous to the DnaB protein in bacteria). Loading of this structure with ORC forms the pre-replicative complex, or the pre-RC and is a key step in the initiation of replication. Initiation won’t occur however untill S-phase cyclin dependent CDK complexes are synthesized and activated. At this point, ATP dissociates and the helicase can perform its activity. This is the committed step and is how replication is regulated during the cell cycle.

Question 24

True or False: All DNA is replicated at the same time during S-phase.

Answer 24

False, more highly condensed chromatin structures usually replicate later in S-phase.

Question 25

How are telomeres synthesized? Describe the final structure of telomeres.

Answer 25

At the end of DNA replication, a telomerase will bind to the ends of the DNA segment. These telomerases have their own RNA primer that will attach to the DNA segment and prep the formation of telomere sequences.
The telomeres loop and feedback into the DNA. It gets tucked in between the two strands.

Question 26

True or False: Telomeres are repetitive DNA sequences attached to the ends of DNA.

Answer 26

False: These are repetitive RNA sequences.