DNA replication

Question 1

Fundamental rules of DNA replication

Answer 1

1) DNA replication is semiconservative (each newly replicated DNA double helix contains one intact parental strand and one newly synthesized daughter strand), 2) Replication begins at an origin and usually proceeds bi-directionally (replication begins at a site of origin and simultaneously moves out in both directions from this point), 3) DNA synthesis always proceeds in a 5’-to-3’ direction

Question 2

Replication forks

Answer 2

are the sites at which DNA synthesis is occurring. First, origin binding proteins recognize and bind to origins of replication. Next, the parental strands of DNA separate and the helix unwinds ahead of the replication fork by helicases.While helicases unwind the double helix, single-strand binding proteins bind to each single strand of DNA and hold it in a single-stranded conformation.

Question 3

origin of replication

Answer 3

are AT rich sequences. The bacterial genome has one origin of replication, humans have 100s of origins of replication on each chromosome.

Question 4

“Problems” that must be overcome for DNA polymerases to copy DNA

Answer 4

1) Unwinding. DNA polymerases are unable to melt duplex DNA in order to separate the two strands that are to be copied. 2) Primer. DNA polymerases can only elongate a pre-existing DNA or RNA strand (the primer); they are unable to initiate a chain de novo. 3) Polarity. The two strands in the DNA duplex are opposite in chemical polarity, but all DNA polymerases catalyze nucleotide addition at the 3’-hydroxyl end of a growing chain, so strands can grow only in the 5’-to-3’ direction.

Question 5

Overview of steps in DNA replication

Answer 5

1) Recognition of replication origin by origin binding proteins 2) DNA melting/unwinding by DNA helicase 3) Relaxation of torsional stress ahead of the replication fork by topoisomerase / gyrase 4) Protection of unwound single-stranded DNA by single stranded binding proteins (SSB) 5) Synthesis of RNA primer by primase 6) Elongation of DNA from the RNA primer by DNA Pol III 7) Removal of RNA primer and copying into DNA by DNA Pol I 8) Ligation of DNA fragments by DNA ligase

Question 6

Topoisomerases

Answer 6

act to prevent the extreme supercoiling of the parental helix that would result as a consequence of unwinding at a replication fork. It break and rejoin DNA chains to relieves torsional strain generated by DNA unwiding.

Question 7

DNA gyrase

Answer 7

a topoisomerase (topo II) inhibited by quinolones, is found mostly in prokaryotes, relieves torsional strain ahead of replication fork by breaking and rejoining double strand DNA.

Question 8

Topo 1

Answer 8

cut one strand of double-stranded DNA, relax the strand, and reanneal the strands.

Question 9

quinolones

Answer 9

a family of synthetic broad-spectrum antibacterial drugs. selectively inhibit the topoisomerase II ligase domain, leaving the two nuclease domains intact. This modification, coupled with the constant action of the topoisomerase II in the bacterial cell, leads to DNA fragmentation via the nucleasic activity of the intact enzyme domains.

Question 10

Replication origins

Answer 10

Replication origins, regardless of organism, 1) unique DNA segments with multiple short repeats, 2) recognized by multimeric origin-binding proteins, 3) usually rich in an A=T base pairs.

Question 11

DnaA

Answer 11

a protein that activates initiation of DNA replication in prokaryotes. It is a replication initiation factor which promotes the unwinding of DNA at oriC. When DNA replication is about to commence, DnaA occupies the AT-rich region, which denatures the DNA and allows for the recruitment of DnaB (helicase)

Question 12

DNA polymerase

Answer 12

catalyze the synthesis of DNA by adding deoxyribonucleotides to the 3’- hydroxyls of the RNA primers and subsequently to the ends of the growing DNA strands. Prokaryotic DNA replication is carried out by two DNA polymerases: DNA Pol I and Pol III. Eukaryotic DNA replication requires at least three DNA polymerases, Pol α, Pol δ, and Pol ε.

Question 13

DNA helicase

Answer 13

They are motor proteins that move directionally along a nucleic acid phosphodiester backbone, separating two annealed nucleic acid strands (i.e., DNA, RNA, or RNA-DNA hybrid) using energy derived from ATP hydrolysis.

Question 14

single stranded binding proteins (SSB)

Answer 14

binds to single-stranded regions of DNA to prevent premature annealing, to protect the single-stranded DNA from being digested by nucleases, and to remove secondary structure from the DNA to allow other enzymes to function effectively upon it. Single-stranded DNA is produced during all aspects of DNA metabolism: replication, recombination and repair.

Question 15

Replication protein A (RPA)

Answer 15

a protein that binds to single-stranded DNA in eukaryotic cells.[1] During DNA replication, RPA prevents single-stranded DNA (ssDNA) from winding back on itself or from forming secondary structures. This keeps DNA unwound for the polymerase to replicate it.RPA also binds to DNA during the Nucleotide Excision Repair process. This binding stabilizes the repair complex during the repair process. A bacterial homolog is called single-strand binding protein (SSB).

Question 16

primase

Answer 16

an enzyme involved in the replication of DNA. DNA primase is, in fact, a type of RNA polymerase which creates an RNA primer (later this RNA piece is removed by a 5’ to 3’ exonuclease); next, DNA polymerase uses the RNA primer to replicate ssDNA

Question 17

DNA Pol III

Answer 17

the major replicative enzyme because it has a sliding clamp that keeps it attached to the DNA template over a long distance. Thus, DNA Pol III has much higher processivity than DNA Pol I.

Question 18

DNA clamp

Answer 18

also known as a sliding clamp, a critical component of the DNA polymerase III holoenzyme, the clamp protein binds DNA polymerase and prevents this enzyme from dissociating from the template DNA strand. The clamp-polymerase protein–protein interactions are stronger and more specific than the direct interactions between the polymerase and the template DNA strand; because one of the rate-limiting steps in the DNA synthesis reaction is the association of the polymerase with the DNA template, the presence of the sliding clamp dramatically increases the number of nucleotides that the polymerase can add to the growing strand per association event. The DNA clamp fold is an α+β protein that assembles into a multimeric structure that completely encircles the DNA double helix as the polymerase adds nucleotides to the growing strand.

Question 19

DNA Pol I

Answer 19

performs clean-up function during DNA replication and repair. DNA Pol I mediates replacement of RNA primers with DNA through its 5’-to-3’ exonuclease activity and 5’-to-3’ DNA polymerase activity.

Question 20

DNA ligase

Answer 20

the Okazaki fragments are joined by DNA ligase, an enzyme that catalyzes formation of phosphodiester bonds between a 3’-hydorxyl group and a 5’-phosphate group of two polynucleotide chains. ATP is the cofactor for
ligase in eukaryotes. NAD+ is the cofactor for ligase in prokaryotes

Question 21

DNA Pol α holoenzyme

Answer 21

Pol α has limited processivity and lacks 3′ exonuclease activity for proofreading errors. Thus it is not well suited to efficiently and accurately copy long templates (unlike Pol Delta and Epsilon). Instead it plays a more limited role in replication. Pol α is responsible for the initiation of DNA replication at origins and during lagging-strand synthesis of Okazaki fragments.

Question 22

DNA Pol δ

Answer 22

synthesizes the lagging strand DNA replication

Question 23

DNA Pol ε

Answer 23

synthesizes the leading strand DNA replication

Question 24

RNA primer

Answer 24

(~10 nucleotides) is formed by copying of the parental DNA strand in a reaction catalyzed by primase, a DNA-dependent RNA polymerase.

Question 25

DNA parental (template) strands

Answer 25

are copied simultaneously at replication forks, although they run in opposite directions.

Question 26

leading strand

Answer 26

formed by continuous copying of the parental strand that runs 3’-to- 5’ toward the replication fork.

Question 27

lagging strand

Answer 27

is formed by discontinuous copying of the parental strand that runs 3’ to 5’ away from the replication fork.

Question 28

Okazaki fragments

Answer 28

As more of the helix is unwound, synthesis of the lagging strand begins from another primer. The short fragments (100-200 bp in eukaryotes and 1000-2000 bp in prokaryotes) formed by this process are known as Okazaki fragments. Leading and lagging strands are synthesized concurrently via looping of the lagging strand

Question 29

exonuclease

Answer 29

The RNA primers are removed by 5’-3’ exonuclease (DNA Pol I in E. coli), then the resulting gaps are filled with the appropriate deoxyribonucleotides, also by DNA Pol I in E. coli. The E. coli DNA Pol I possesses three activities: (1) a 5’-to-3’ DNA polymerase activity requiring a 3’-OH primer and a DNA template strand; (2) a 5’-to-3’ exonuclease activity for RNA primer removal; (3) a 3’-to-5’ exonuclease activity for proofreading.

Question 30

Fidelity of replication

Answer 30

is very high with an overall error rate of 10-9 to 10-10 because 1) Polymerases discriminate between the correct and incorrect nucleotides to incorporate based on the ability to form hydrogen bonds between the complimentary bases A-T and G-C AND the common geometry of the A-T and G-C base pairs that allow them to fit into the active site of the polymerase. Together these account for accuracy of replication such that 1 wrong nucleotide is incorporated per 10,000-100,000 correct nucleotides. 2)Errors (insertion of an inappropriate nucleotide) that occur during replication can be corrected by proofreading during the replication process. 3) Post-replicational repair processes

Question 31

DNA polymerase proofreading

Answer 31

When an incorrect base pair is recognized, DNA polymerase moves backwards by one base pair of DNA. The 3’-5’ exonuclease activity of the enzyme allows the incorrect base pair to be excised. Proof reading increases the accuracy of replication by 100-1,000 fold such that 1 wrong nucleotide remains per million to 100 million correct nucleotides.

Question 32

processive DNA synthesis

Answer 32

an enzyme’s ability to catalyze “consecutive reactions without releasing its substrate”. For example, processivity is the average number of nucleotides added by a polymerase enzyme, such as DNA polymerase, per association/disassociation with the template. DNA polymerases associated with DNA replication tend to be highly processive, while those associated with DNA repair tend to have low processivity. Because the binding of the polymerase to the template is the rate-limiting step in DNA synthesis, the overall rate of DNA replication during S phase of the cell cycle is dependent on the processivity of the DNA polymerases performing the replication. DNA clamp proteins are integral components of the DNA replication machinery and serve to increase the processivity of their associated polymerases.

Question 33

distributive DNA synthesis

Answer 33

is distributive if it dissociates from the primer‐template after each incorporation of a single nucleotide.

Question 34

Proliferating cell nuclear antigen (PCNA)

Answer 34

a DNA clamp that acts as a processivity factor for DNA polymerase δ in eukaryotic cells and is essential for replication. PCNA is a homotrimer and achieves its processivity by encircling the DNA, where it acts as a scaffold to recruit proteins involved in DNA replication, DNA repair, chromatin remodeling and epigenetics.

Question 35

b-subunit

Answer 35

the sliding clamp of prokaryotic DNA Pol III

Question 36

Clamp loader complex

Answer 36

Also called replication factor C, its role as clamp loader involves catalysing the loading of PCNA on to DNA. It binds to the 3’ end of the DNA and uses ATP to open the ring of PCNA so that it can encircle the DNA. ATP hydrolysis causes release of RFC, with concomitant clamp loading onto DNA. Leading and lagging strands are synthesized
concurrently via looping of the lagging strand

Question 37

replisome

Answer 37

a complex molecular machine that carries out replication of DNA. In terms of structure, the replisome is composed of two replicative polymerase complexes, one of which synthesizes the leading strand, while the other synthesizes the lagging strand. The replisome is composed of a number of proteins including helicase, RFC, PCNA, gyrase/topoisomerase, SSB/RPA, primase, DNA polymerase I, RNAse H, and ligase. in prokaryotics, Both leading and lagging strands are synthesized by DNA Pol III, a multimeric holoenzyme with two identical core polymerase complexes.

Question 38

Reverse transcription

Answer 38

catalyzes synthesis of DNA from an RNA template, include retroviruses and telomerase

Question 39

Retroviruses

Answer 39

contain RNA as their genetic material. The retroviral RNA serves as a template for synthesis of DNA by reverse transcriptase.

Question 40

Telomerase

Answer 40

an RNA-dependent DNA polymerase that maintains chromosomal ends by copying the telomeric repeat sequence from an RNA template, in order to restore the ends of chromosomes (telomeres) in human cancer and stem cells. . Telomerase activity is repressed in normal somatic cells. In cancer cells, the telomerase enzyme is de-repressed, restoring the ends of the chromosomes to their full length and therefore blocking the normal cell death of old cells, promoting tumor growth. As such, telomerase is a potential target for anti-cancer drugs. stem cells also have increased telomerase activity.

Question 41

end replication problem

Answer 41

refers to the fact that the leading stand can be synthesized to the very end, but the lagging strand cannot during DNA replication. This is because you need an RNA primer to begin synthesis of each piece of the lagging strand DNA, but at the end of the DNA there is nothing for this piece to attach to thus the last section of the lagging strand cannot be synthesized. The result is that the telomeres (i.e. the end of chromosomes) get shorter and shorter as a cell replicates its genomes and divides, until they are so short that they signal for cell death – a normal healthy process in ageing cells.

Question 42

azidothymidine (AZT)

Answer 42

drug used to delay development of AIDS (acquired immunodeficiency syndrome) in patients infected with HIV (human immunodeficiency virus). AZT works by selectively inhibiting HIV’s reverse transcriptase, the enzyme that the virus uses to make a DNA copy of its RNA.

Question 43

Aciclovir

Answer 43

a guanosine analogue antiviral drug. It is one of the most commonly used antiviral drugs, that is primarily used for the treatment of herpes simplex virus infections, as well as in the treatment of varicella zoster (chickenpox) and herpes zoster (shingles). inhibits and inactivates HSV-specified DNA polymerases preventing further viral DNA synthesis without affecting the normal cellular processes.

Question 44

Dyskeratosis congenita (DKC)

Answer 44

is a rare progressive congenital disorder that in some ways resembles premature aging (similar to progeria). The disease mainly affects the integumentary system (i.e, the skin, the organ system that protects the body from damage), with a major consequence being anomalies of the bone marrow. Though the exact pathology of the disease is not yet fully understood, most evidence points to dyskeratosis congenita being primarily a disorder of poor telomere maintenance.