Lecture 14: Replication and Transcription - Biomed

What is the structure of the chromosome in bactera?

Closed circular DNA molecule

The bacteria chromosome resides in a region of the cell known as the _____________.

Nucleoid

_____________________ is the coiling of the DNA axis upon itself

Supercoiling

Bacterial chromosome is arranged into ____________ and is ________-handed and _____________ supercoiled

- Loops

- Right

- Negatively

Why must eukaryotic DNA be packed?

Packing of eukaryotic DNA is required for all of it to fit in the nucleus

What is chromatin?

DNA + Protein

DNA in the chromatin is very tightly associated with proteins called ____________, which package and order the DNA into structural units called ___________________.

- histones

- nucleosomes

_______________ are small basic proteins which bind tightly to DNA

Histones

What are the 5 types of histones?

H1, H2A, H2B, H3, H4

___________________ are the Fundamental Organizational Units of Chromatin. Histone + DNA.

Nucleosomes

The nucleosomes make the chromatin structure look like....

Beads on a string

Nucleosomes structures condense into a _____nm filament

30 nm

Chromatin is further condenses and folds in a zig-zag pattern into a filament which is _______nm in diameter. This filament is a _________ consisting of ___ nucleosomes per turn.

- 300 nm

- solenoid

- 6

Which histone stabilizes the solenoid structure?

H1 histones

The 300 nm filaments form ________ which are attached to a protein scaffold giving rise to condensed _____________ chromosomes.

- loops

- metaphase

Human chromosomes contain _____ chromosomes (or _____ pairs of chromosomes)

- 46

- 23

What are the two types of human cells that don't have 46 chromosomes, and how many chromosomes do they have?

- Germ cells (23 chromosomes)

- Red blood cells (no chromosomes)

Diploid human cells contain ____ chromosomes

46

Haploid human cells contain ____ chromosomes

23

Out of 23 pairs: 22 pairs are called ____________ and a pair of _____ chromosomes (XX: Female or XY: Male)

- autosomes

- sex

The mechanism of DNA replication has been well studied in the bacterium _____________.

E. coli

DNA Synthesis replication in Prokaryotes serves as a model to explain the more ____________ DNA replication in _________________ organisms

- Complex

- Eukaryotic

Why must cells synthesize new DNA when dividing?

Whenever a cell divides, the two new daughter cells must contain the same genetic information, or DNA, as the parent cell.

What are 3 processes/examples in humans that require cellular division?

1. Renewal of skin

2. Wound healing

3. Growth of Embryo to Fetus

What are the 3 steps in replication?

1. Initiation

2. Elongation

3. Termination

Where does DNA replication begin on the chromosome?

Replication begins at the origin of replication (Ori C)

What properties of Ori C aid in initiating DNA replication?

Ori C has A-T rich region; thus, has fewer Hydrogen bonds than G-C region helping to "melt" DNA

What is the initiator protein, and what does it do?

- The initiator protein is DnaA

- DnaA binds to OriC to intiate replication

What 3 enzymes/proteins are involved with the unwinding and stabilization of DNA?

- Helicase

- Gyrase

- SSB Proteins (Single Strand Binding Proteins

TRUE/FALSE

DNA replicates unidirectionally

False, DNA replicates bidirectionally

Which enzyme/protein unwinds and separates the parental DNA strands?

DNA Helicase (DnaB)

Which enzyme/protein associates with the single strands and prevents them from re-associating with one another. Also protects the single strands from cleavage by endonucleases.

Single Stand Binding Proteins (SSB)

Which enzyme/protein relieves stress generated by unwinding of DNA by the Helicase in front of the replication fork?

DNA Gyrase (Topoisomerase)

How does Topoisomerase relieve stress from unwinding DNA?

Topoisomerase creates a tiny cut in a single strand of DNA to allow the second strand to unwind itself and then ligates this break in the strand

How many DNA polymerases exist in E. coli? What are their names?

- 3

- DNA Pol I, II, and III

Which Prokaryotic DNA polymerase is the major enzyme involved in DNA replication?

DNA Polymerase III

Which Prokaryotic DNA Polymerase is involved in RNA primer removal and filling up the resulting gap?

DNA polymerase I

Which Prokaryotic DNA Polymerase is involved in DNA repair?

DNA Polymerase II

Which Prokaryotic DNA Polymerase(s) has/have 3'-5' exonuclease activity

DNA Polymerase I, II, and III

Which Prokaryotic DNA Polymerase(s) has/have 5'-3' exonuclease activity as well?

DNA Polymerase I only

Can DNA polymerase add nucleotides directly onto the DNA strand?

No, the enzyme that adds nucleotide to elongate DNA cannot initiate the process from scratch (de novo synthesis)

What is de novo synthesis?

The production of complex substances from simple molecules

What does Primase do?

- Synthesize RNA primer

- Allows DNA polymerase to synthesize DNA starting from RNA primer

How long is a RNA primer?

~5-10 nucleotides long

_______ (DNA or RNA) Primase can synthesize RNA primers

DNA Primase

DNA primase has very low ___________ and hands over elongation to DNA ______________.

- fidelity (accuracy)

- polymerase

The replication fork, DNA polymerase III, Leading strand, and Lagging strand are all parts of the ______________ phase of DNA synthesis.

Elongation

DNA is always synthesized from ___' to ___'

5' to 3'

What is the site of simultaneous unwinding and synthesis?

Replication Fork

What enzyme catalyzes elongation of DNA?

DNA polymerase III

Which strand involves the continuous synthesis of new DNA 5' to 3' direction?

Leading strand

In the leading strand, the template strand is ___' to ____', and the newly synthesized strand is made from ____' to ____'

- Template: 3' to 5'

- Newly synthesized: 5'- 3'

Which strand is the discontinuous synthesis in the 5' to 3' direction?

Lagging strand

In the lagging strand, the template strand is ___' to ____', and the newly synthesized strand is made from ____' to ____'

- Template: 5' to 3'

- Newly synthesized: 5' to 3'

What is the complication of the lagging strand?

The discontinuous synthesis requires multiple primers to initiate replication

________________ fragments are found in the lagging strand, and the short fragments are then _____________ together to make a continuous DNA strand.

- Okazaki

- ligated

How many nucleotides are in the short fragments of the lagging strand in prokaryotes?

1-2000 nucleotides

How many nucleotides are in the short fragments of the lagging strand in eukaryotes?

~200 nucleotides

The end result of the lagging strand is overall ___' to ___' synthesis

5' to 3'

Eventually, the two replication forks of the circular E. coli chromosome meet at a ____________ region: Leading to ____________ of replication

- terminus

- termination

Opposing replication forks ______ when they collide

halt

TRUE/FALSE

RNA primers are removed and filled by DNA polymerase II

FALSE

RNA primers are removed and filled by DNA polymerase I

What feature of DNA polymerase I allows it to remove and fill RNA primers?

5' to 3' exonuclease activity

TRUE/FALSE

DNA polymerase I joins the ends of adjacent Okazaki fragments?

FALSE

Which enzyme joins together Okazaki fragments?

DNA ligase

What bond does DNA ligase form between the flanking nucleotides?

Phosphodiester bond

What are the 3 features of DNA Replication?

- Semi-conservative

- Always in 5' to 3' direction

- Semi-discontinuous

How are DNA strands semiconservative?

Each new DNA molecule consists of an old and new strand

How are DNA molecules semi-discontinuous?

Leading strand continuous and lagging strand discontinuous

What are the 3 characteristics of DNA Polymerase III?

- Proofreading

- Processivity

- High fidelity

How is DNA polymerase III capable of proofreading?

Possess a 3' to 5' exonuclease activity

How does DNA Polymerase III have processivity?

- Processivity is the amount of nucleotides DNA adds during a single binding event

- Synthesizes DNA strand continuously without dissociating from the template (leading strand).

How does DNA polymerase III have high fidelity?

Has low error rate in incorporating nucleotides during DNA replication

TRUE/FALSE

The basic mechanism of prokaryotic DNA replication is the same as that of eukaryotes.

TRUE

Eukaryotic contains several polymerases but ____________ functions to E. coli DNA polymerases

Analogous

_____ different DNA polymerases exist in Eukaryotic organisms

- 16

What eukaryotic DNA polymerase is involved with primase activity?

DNA polymerase ⍺

What eukaryotic DNA polymerase is involved with elongation activity?

DNA polymerase 𝝳

Eukaryotic DNA replication is restricted to ____ of cell cycle

S

The first way eukaryotic DNA replication differs from prokaryotes is that eukaryotes have multiple replication of origin called ________.

ARS (autonomously replicating sequences)

The second way eukaryotic DNA replication differs from prokaryotes is that eukaryotes have a rate of replication that is __________ than prokaryotes.

- slower

What is the rate of replication in eukaryotes and prokaryotes?

- Eukaryotes: ~50 nucleotides/second

- Prokaryotes: ~500 nucleotides/second

The third difference between eukaryotic and prokaryotic DNA replication is that eukaryotes have _________ chromosomes, which creates of problem. The presence of ___________ can lead to strand shortening.

- Linear

- Telomeres

What is the end replication problem?

- 3'-end of lagging strand presents unique problem

- No place for RNA primer to be synthesized at end of the DNA

What is the solution to the end replication problem?

Telomere Replication

Ends of DNA have unique sequences known as ______________ which interact with an enzyme, ______________.

- Telomeres

- Telomerase

In Telomere replication, RNA serves as template to add _________________ repeats

- Hexanucleotide

How does telomerase recognize the parent strand?

Recognizes the parent strand due to presence of the telomere sequence

What is the telomere sequence in humans?

TTAGGG for humans

What does Telomerase do?

- Extends parent DNA strand in the 5' to 3' direction using the RNA template. (Adds several hexanucleotide repeats)

- These repeats provide a template for the Primase and the DNA Pol to synthesize the remaining Okazaki fragment

What enzyme uses a single strand of RNA as a template to synthesis DNA?

Reverse transcriptase

The synthesized DNA by reverse transcriptase is known as ______________ DNA or _______ DNA (__DNA).

- Complementary

- Copy

- cDNA

What uses a reverse transcriptase to copy the viral RNA genome to cDNA?

Retrovirus (RNA containing virus)

TRUE/FALSE

The cDNA produced by retroviruses will integrate into the host genome

TRUE

- Ex. HIV

RNA exists as a __________ strand but can form ____________ structures within the chain (Stem-loop structures).

- single

- secondary

RNA consists of a polynucleotide chain held together by ___' to ___' ____________________ bonds.

- 3' to 5'

- phosphodiester

What are the nitrogenous bases in RNA?

- A, C, G, U

Which nitrogenous base replaces Thymine?

Uracil

What is the pentose sugar in RNA?

Ribose, instead of deoxyribose

Which Hydrogen from deoxyribose sugar is replaced by OH in ribose sugars?

Hydrogen is replaced by OH group at 2' position of the sugar