Molecular biology basics

Question 1

What is a nucleoside?
What is a nucleotide?

Answer 1

Nucleoside = base + sugar
Nucleotide= base, sugar and phosphate

base joins on carbon 1
phosphate group joins on carbon 5 of the sugar

when a new nucleotide joins another nucleotide the phosphate O- joins with OH on 3rd carbon of the sugar this happens last DNA replication is 5’ to 3’ so you would read it CAG if it was 3 nucleotide

Question 2

What are the similarities and differences DNA vs RNA?

Answer 2

DNA (stable) (has only H on 2nd carbon)
Base composition= A, T, G, C
Chain=double-stranded
Number of molecules per cell= few
functions=information storage

RNA (more unstable) (more unstable because of extra Oxygen on 2’ carbon. gets degraded quickly) (has OH on 2nd carbon)
Base composition= A, U, G, C
Chain=single-stranded (can have double stranded regions)
Number of molecules per cell= many
functions= many different functions (transport of
information, structural, enzymatic, )

Question 3

Why did they win the nobel prize (DNA )

Answer 3

nucleoside modifications

Question 4

Name the purines and the pyrimidines?

Answer 4

purines= adenine and guanine
pyrimidines= uracil, thymine and cytosine

Question 5

Why is RNA more unstable?

Answer 5

The extra oxygen on 2’ carbon which makes it much more reactive

Question 6

Explain RNA world

Answer 6

RNA can store information but also has enzymatic activity so theory= there was RNA and it started to code for proteins but its not very stable so DNA then was created as it is more stable and better for holding information.

Question 7

What do ribosomes have

Answer 7

proteins and rRNA

Question 8

Why are reverse transcriptase used and where are they normally found?

Answer 8

RNA is very reactive so retro transcribing RNA into DNA allows for the sequence to be the same just transcribed but to also be stored. They use reverse transcriptases. They are found in viruses mostly

Question 9

What is the difference between a cDNA and the original DNA?

Answer 9

Introns aren’t included in cDNA only contains the expressed part of the gene

Question 10

What is gDNA?

Answer 10

genomic DNA it is chromosomal DNA

Question 11

describe gDNA, mRNA and cDNA

Answer 11

gDNA=(this includes everything
mRNA =no introns, only certain genes here from gDNA
cDNA =mRNA transcribed back to DNA so only summary of gDNA

Question 12

How is DNA replicated?

Answer 12

semi-conservative and semi-discontinuous

Question 13

What is semi-discontinuous and describe how DNA replicates?

Answer 13

A mode of DNA replication in which one new strand is synthesized continuously, while the other is synthesized discontinuously as Okazaki fragments.

polymerisation starts in parental molecule, it opens up and gets synthesised, becomes leading strand.
Problem on other side

Two molecules start coming apart double strand open ups, 5’ to 3’ direction
The leading strand gets replicated in one large fragment in this direction however the lagging strand get replicated in smaller fragments because DNA polymerase can only add nucleotides from the 3’ end.
DNA helicase opens strands exposing templates. RNA primer assembled in 5’ to 3’ by primase. DNA polymerase attaches. There is a space at the replication fork so when it unwinds more leading strand can carry on but lagging strand has to make another RNA primer.
On the lagging strand it has various fragments called okazaki fragments, another DNA polymerase removes RNA primers and replaces it with DNA. Finally, DNA ligase joins the two strands

Question 14

What do we need to know for DNA replication and the issues?

Answer 14

• where to start? how to continue? how to terminate?
• torsional strain from unwinding the double helix (helicase + topoisomerase), damage (nicks) while unwinding DNA molecule
• DNA strands are anti-parallel (primase, ligase)
• needs “fidelity” (DNA pol III, DNA pol I)
  => three main phases (initiation, elongation, termination) and multiple enzymes

Question 15

DNA replication in bacteria - Initiation

Answer 15

Bacteria have circle of chromosomes, one place where replication starts called OriC in e.coli

they have lot more A and T’s which means there are less hydrogens so less energy is required for it to be opened up

Question 16

Describe DNA replication in bacterial cells summary

Answer 16

Summary
* A single initiation point (OriC in E. coli)
* Main initiation proteins: DnaA (initiator), DnaB (helicase),
DnaC (loader), DnaG (primase)
* Replication occurs at replication fork, is bidirectional and
semi-discontinuous
* Lagging strand is synthesised via Okazaki fragments
* DNA Pol III Holoenzyme synthesises both strands (5’→3’)
* DNA Pol I replaces RNA primers on lagging strand with DNA
* DNA ligase fills the gaps

Question 17

What is an endonuclease vs exonuclease

Answer 17

exonuclease remove nucleotides at the end
Endonuclease cuts in the middle

Question 18

What are the properties of the DNA polymerases

Answer 18

1. DNA Polymerase I (Pol I):

Function: Pol I plays a multifaceted role in DNA metabolism, including DNA replication and repair.
5’ to 3’ Polymerase Activity: It can synthesize DNA in the 5’ to 3’ direction, adding nucleotides to a growing DNA strand.
5’ to 3’ Exonuclease Activity: Pol I has 5’ to 3’ exonuclease activity, which allows it to proofread and correct errors in the newly synthesized DNA.
3’ to 5’ Exonuclease Activity: This enzyme also has 3’ to 5’ exonuclease activity, which allows it to remove RNA primers during DNA replication and replace them with DNA.
Role in Lagging Strand: In bacterial DNA replication, Pol I is responsible for processing the Okazaki fragments on the lagging strand. It removes RNA primers and fills the gaps with DNA nucleotides.
Repair Functions: In DNA repair processes, Pol I is involved in base excision repair and nucleotide excision repair, where it replaces damaged or incorrect nucleotides with the correct ones.
2. DNA Polymerase II (Pol II):

Function: Pol II primarily serves as a backup or repair polymerase.
5’ to 3’ Polymerase Activity: Like other DNA polymerases, it can synthesize DNA in the 5’ to 3’ direction.
Lacks Exonuclease Activity: Pol II lacks significant exonuclease activity and has a lower proofreading capability compared to Pol I and Pol III.
Error-Prone DNA Repair: It is often employed during DNA repair processes where fidelity is not a priority, as it tends to be less accurate compared to other polymerases.
Backup Polymerase: In E. coli, Pol II may be used when other polymerases encounter obstacles during DNA replication.
3. DNA Polymerase III (Pol III):

Function: Pol III is the main replicative polymerase in bacterial DNA replication.
High Processivity: Pol III is highly processive, meaning it can add numerous nucleotides to a growing DNA strand before dissociating from the template.
5’ to 3’ Polymerase Activity: It synthesizes DNA in the 5’ to 3’ direction, adding nucleotides to the growing DNA strand.
3’ to 5’ Exonuclease Activity: Pol III has 3’ to 5’ exonuclease activity, which allows it to proofread and correct errors during DNA replication.
Part of Holoenzyme: In the bacterial replication machinery, Pol III functions as part of a larger complex known as the Pol III holoenzyme, which includes multiple subunits with different functions.
Leading Strand Synthesis: It synthesizes the leading strand during DNA replication continuously.

Question 19

Explain in depth bacterial DNA replication

Answer 19

1. Initiation:
   DnaA Protein(initiator): DnaA plays a central role in initiating replication. It recognizes the DnaA-boxes within the origin of replication (OriC) and binds to them. When bound to ATP, DnaA forms complexes that promote DNA strand separation (DnaA-ATP). This results in the unwinding of the DNA strands at OriC, creating single-stranded DNA (ssDNA) regions.
2. Helicase Loading:
   DnaC Protein: DnaC(loader) helps load the DnaB helicase onto the ssDNA regions created by DnaA. DnaB is an essential helicase that unwinds the DNA double helix by breaking hydrogen bonds between base pairs.
3. Primase and RNA Primer Synthesis:
   DnaG (Primase): DnaG synthesizes short RNA primers complementary to the exposed ssDNA. These primers serve as starting points for DNA synthesis. DnaG binds to DnaB, forming a complex known as the primosome (DnaG + DnaB). This primosome is critical for the synthesis of RNA primers, which are necessary for the initiation of DNA synthesis.
4. DNA Polymerases:
   DNA Polymerase III: This is the primary replicative polymerase. It adds deoxyribonucleotides to the 3’ end of the RNA primer or the existing DNA strand, extending the new DNA strand in the 5’ to 3’ direction.
   DNA Polymerase I: DNA Polymerase I has both polymerase and 5’ to 3’ exonuclease activities. After DNA Polymerase III synthesizes an Okazaki fragment on the lagging strand, DNA Polymerase I removes the RNA primer and replaces it with DNA nucleotides.
5. Leading and Lagging Strand Synthesis:
   The leading strand, which is synthesized continuously in the 5’ to 3’ direction, is synthesized by DNA Polymerase III as it moves towards the replication fork.
   The lagging strand is synthesized discontinuously in Okazaki fragments.
   * Okazaki Fragment Synthesis:
   DNA polymerase III, the primary replicative polymerase, works on the lagging strand in a discontinuous manner.It starts from the RNA primer and adds DNA nucleotides in the 5’ to 3’ direction, creating the first Okazaki fragment.
   DNA polymerase III can only synthesize DNA in the 5’ to 3’ direction, so it moves away from the replication fork. As the replication fork continues to open, more single-stranded DNA is exposed.
   Primase synthesizes a new RNA primer further down the lagging strand, and DNA polymerase III synthesizes another Okazaki fragment.
   This process continues, creating a series of Okazaki fragments along the lagging strand, each initiated by a separate primer.
   * RNA Primer Removal and DNA Replacement:

After DNA polymerase III has completed each Okazaki fragment, DNA polymerase I takes over.
DNA polymerase I has both 5’ to 3’ polymerase activity and 5’ to 3’ exonuclease activity.
First, it uses its exonuclease activity to remove the RNA primer and replace it with DNA nucleotides. This leaves a small gap between adjacent Okazaki fragments.
* Okazaki Fragment Joining:
DNA ligase, an enzyme, is responsible for sealing the nicks between the adjacent Okazaki fragments.
It catalyzes the formation of a phosphodiester bond between the 3’ end of one DNA fragment and the 5’ end of the next, creating a continuous strand on the lagging strand.
The result of this process is a newly synthesized DNA strand on the lagging strand that is composed of a series of Okazaki fragments joined together by DNA ligase. This newly synthesized lagging strand is complementary to the template strand of the original DNA.
In contrast, the leading strand is synthesized continuously in the 5’ to 3’ direction as the replication fork advances, allowing DNA polymerase III to continuously add nucleotides. The lagging strand, due to its discontinuous nature, requires the synthesis of multiple primers and the subsequent joining of Okazaki fragments to complete the process.
7. Termination:
Termination of replication occurs when two replication forks meet on the opposite side of the circular chromosome from the origin (Ter site).

Question 20

What are the differences between bacterial and eukaryotic genomes

Answer 20

DNA replication
Differences between bacterial and eukaryotic genomes
* circular vs linear
* small vs large
Structural characteristics and differences affecting how DNA is replicated.
Initiation is more complex in eukaryotes
* Eukaryotic chromosomes&raquo_space;> E. coli chromosome
* Eukaryotic polymerases slower than bacterial ones
* What does this mean for DNA replication?
* Eukaryotes have multiple origins on a single chromosome (human chromosomes ~ 10,000 origins compared to 1 in ecoli)
* Origins of replication are regulated => they “start” once per each cell
cycle (cyclin-CDKs

Question 21

What are the steps of the cell cycle in eukaryotic cells

Answer 21

1. G1 Phase (Gap 1):
   In this phase, the cell grows and carries out its normal functions.
   It prepares for DNA replication, and it is the stage where cells decide whether to enter the cell cycle or go into a non-dividing state (G0).
2. S Phase (Synthesis):
   DNA synthesis occurs in this phase. The cell replicates its DNA to ensure that the two resulting daughter cells will each receive a complete set of genetic information.
   By the end of the S phase, the cell has two complete sets of chromosomes, referred to as sister chromatids, attached at the centromere.
3. G2 Phase (Gap 2):
   More growth and preparation for cell division occur during this phase.
   The cell checks for errors in DNA replication and prepares for mitosis or meiosis.
4. M Phase (Mitotic Phase):
   The M phase consists of two major processes:
   Mitosis: This is the process of nuclear division, which ensures that the genetic material is equally distributed between the two daughter cells. Mitosis is divided into several subphases:
   Prophase: Chromatin condenses into visible chromosomes, the nuclear envelope breaks down, and the mitotic spindle forms.
   Metaphase: Chromosomes align at the cell’s equator, known as the metaphase plate.
   Anaphase: Sister chromatids are separated and pulled towards opposite ends of the cell.
   Telophase: Chromatids arrive at the opposite poles, and new nuclear envelopes form around each set of chromosomes.
   Cytokinesis: This is the division of the cytoplasm and other organelles to produce two separate daughter cells. In animal cells, a contractile ring of actin and myosin filaments pinches the cell membrane, creating two distinct daughter cells. In plant cells, a new cell wall forms in the middle, dividing the cell into two.
   After M phase, the cell cycle can either continue with another G1 phase (if the cell is to divide again) or enter a non-dividing state called G0. In G0, cells may remain quiescent, carry out their specialized functions, or await signals to re-enter the cell cycle.

The cell cycle is tightly regulated to ensure the proper progression through each phase and to prevent uncontrolled cell division. Various checkpoints and regulatory proteins, such as cyclins and cyclin-dependent kinases (CDKs), govern the cell cycle and maintain its integrity.

Question 22

Where and what are the checkpoints during the cell cycle?

Answer 22

G1 Checkpoint (Restriction Point):
This checkpoint occurs at the end of the G1 phase, just before the cell enters the S phase.
It checks for favorable conditions, such as nutrient availability, cell size, and DNA damage.
If conditions are appropriate and there is no significant DNA damage, the cell is given the “go-ahead” to proceed to the S phase. If conditions are not suitable, the cell may temporarily arrest or enter a non-dividing state (G0).

G2 Checkpoint:
This checkpoint takes place at the end of the G2 phase, before the cell enters the M phase (mitosis).
It verifies that DNA replication in the S phase is complete and accurate.
If the DNA has been replicated successfully and there are no issues, the cell can proceed to mitosis. However, if DNA damage is detected, the cell cycle may be halted for repair or the initiation of apoptosis (programmed cell death).

Metaphase Checkpoint:
This checkpoint is during the metaphase stage of mitosis.
It ensures that all chromosomes are correctly aligned at the metaphase plate.
The checkpoint ensures that the spindle fibers are correctly attached to the centromeres of the sister chromatids.
If everything is in order, the cell proceeds to anaphase. If not, it delays anaphase until the issues are resolved.

Spindle Assembly Checkpoint:
This checkpoint monitors spindle fiber attachment to kinetochores, structures on chromosomes.
It occurs during both mitosis and meiosis.
The checkpoint ensures that all chromosomes are attached to the spindle fibers before anaphase begins.
If attachment is incomplete or incorrect, the cell delays anaphase.

The cell cycle checkpoints are controlled by various regulatory proteins and signaling pathways, including cyclins and cyclin-dependent kinases (CDKs).

Question 23

Describe telomeres

Answer 23

Linear chromosomes => telomeres
* short DNA repeats (e.g. TTAGGG) (stronger bond)
* G-rich strand + C-rich strand
* every round of replication loses up to 200bp
Telomerases avoid chromosomal shortening
* ribo-nucleo-proteins with reverse transcriptase activity + intrinsic RNA
primer (maintain telomere length and restore 3’ overhang)
* maintain G-rich repeats at end of telomeres without need for complementary template DNA
* frequently activated in cancer cells

Question 24

Describe DNA replication eukaryotic cells?

Answer 24

1.Initiation:
DNA replication begins at specific sites along the DNA called origins of replication. Eukaryotic genomes have multiple origins.
A complex of initiator proteins, including the origin recognition complex (ORC), binds to the origins and unwinds the DNA.
This unwinding allows for the assembly of the pre-replication complex (pre-RC) composed of additional proteins like Cdc6 and Cdt1.
The pre-RC is essential for the loading of the DNA helicase, known as the MCM complex (minichromosome maintenance), which is a key player in unwinding the DNA.

2. DNA Unwinding:
   The DNA helicase, along with other proteins, unwinds the double helix, creating two single-stranded DNA templates for replication.
3. Primase and Primer Synthesis:
   DNA polymerases cannot initiate DNA synthesis without a primer. Primase synthesizes short RNA primers complementary to the single-stranded DNA.
   These primers provide a starting point for DNA synthesis by DNA polymerases.
4. DNA polymerases
   primarily DNA polymerase α (Pol α), DNA polymerase δ (Pol δ), and DNA polymerase ε (Pol ε), synthesize the new DNA strands.
   Pol α initiates synthesis with RNA primers, while Pol δ and Pol ε extend the strands. Pol δ is involved in the lagging strand synthesis, and Pol ε in the leading strand synthesis.
   DNA polymerases add complementary deoxyribonucleotides to the 3’ end of the primer or the existing DNA strand.
   DNA polymerases have 3’ to 5’ exonuclease activity, which allows them to proofread and correct errors during replication.
5. Leading and Lagging Strand Synthesis:
   The leading strand is synthesized continuously in the 5’ to 3’ direction, while the lagging strand is synthesized discontinuously in Okazaki fragments.
   Okazaki fragments are joined together by DNA ligase to create a continuous lagging strand.
6. Termination:
   DNA replication continues until it reaches specific termination sites or sequences.
   The two newly synthesized DNA molecules are released, each consisting of an original strand and a newly synthesized strand.
7. Chromatin Assembly:
   After DNA replication, the new DNA must be properly packaged into chromatin. Histones are involved in this process, and DNA replication-coupled chromatin assembly ensures that the newly replicated DNA is correctly assembled into chromatin.