i want 25 mutiple choice questions with their model answer below each question about the following: Prokaryotic & Eukaryotic Origin of Replication
Multiple origins of replication in eukaryotes
Single origin of replication in prokaryotesDNA Topoisomerases solves the problem of Supercoils
As the 2 DNA strands of the double helix separate, positive supercoils
appear ahead of the replication fork due to overwinding.
DNA topoisomerases are enzymes that cleave one strand of the
double helix, releasing the supercoils.Inhibition of Topoisomerase by Anticancer
Agents
Action of TopoisomeraseDNA synthesis occurs at the replication fork
DNA synthesis occurs at a replication fork, producing new strands termed the
leading strand and the lagging strand.
The “railroad tracks” represent double-stranded DNA. Some of the enzymes
involved in DNA synthesis are shown: DNA B (helicase), DNA primase, DNA
polymerase, and single-strand DNA binding protein (SSBP). The leading strand
is replicated in a continuous fashion.Direction of DNA replication
The DNA polymerases (DNA pols) responsible for copying the DNA templates are only
able to read the parental nucleotide sequences in the 3′→5′ direction, and they
synthesize the new DNA strands only in the 5′→3′ (antiparallel) direction. Therefore,
beginning with one parental double helix, the two newly synthesized stretches of
nucleotide chains must grow in opposite directions, one in the 5′→3′ direction toward
the replication fork and one in the 5′→3′ direction away from the replication fork.The Replication ForkImportant definitions:
1. Leading strand: The strand that is being copied in the direction of the advancing replication fork is
synthesized continuously.
2. Lagging strand: The strand that is being copied in the direction away from the replication fork is
synthesized discontinuously, with small fragments of DNA. These short stretches of discontinuous
DNA, termed Okazaki fragments, are eventually joined (ligated) by ligase to become a single,
continuous strand. The new strand of DNA produced by this mechanism is termed the lagging
strand.
3. RNA primer: DNA pols cannot initiate synthesis of a complementary strand of DNA on a totally
single-stranded template. Rather, they require an RNA primer, which is a short piece of RNA base-
paired to the DNA template, thereby forming a double-stranded DNA-RNA hybrid. The free hydroxyl
group on the 3'-end of the RNA primer serves as the first acceptor of a deoxynucleotide by action
of a DNA polHowever, for the lagging strand, RNA primers are periodically added by the DNA
primase along the strand, they are short stretches of RNA (~10 nucleotides long) that
are complementary and antiparallel to the DNA template, these short RNA sequences
are constantly being synthesized at the replication fork on the lagging strand, but only
one RNA sequence at the origin of replication is required on the leading strand. DNA
polymerase III elongates these RNA primers to form Okazaki fragments. When the
Okazaki fragment is complete, the DNA polymerase III on the lagging strand will shift
to the next RNA primer to initiate another Okazaki fragment.
The exonuclease activity of DNA polymerase I removes the RNA primers and replaces
them with DNA. DNA ligase seals the gaps in the DNA strands to complete synthesis
of the lagging strand.When the 3′ end of the elongating Okazaki fragment reaches the 5′ end of the
previously synthesized fragment, DNA polymerase III releases the template
and finds another RNA primer.
Farther back along the lagging strand, synthesizing another Okazaki fragment.
Eventually, the Okazaki fragments are joined by DNA polymerase I. This
enzyme, which also has a role in DNA repair, has an exonuclease activity that
permits it to remove and replace a stretch of nucleotides as it proceeds along a
DNA template. During DNA replication, DNA polymerase I removes the RNA
primer and replaces it with DNA. Finally, DNA ligase joins the lagging-strand
DNA fragments to form a continuous strand.Termination
Replication termination in E. coli is mediated by sequence-specific binding of the
protein Tus (terminus utilization substance) to replication termination (ter) sites on the
DNA, stopping the movement of the replication fork.Eukaryotic DNA Replication
• The process of eukaryotic DNA replication closely follows that of prokaryotic
DNA synthesis. Some differences, such as the multiple origins of replication in
eukaryotic cells versus single origins of replication in prokaryotes. Eukaryotic
origin recognition proteins, ssDNA-binding proteins, and ATP-dependent DNA
helicases have been identified, and their functions are analogous to those of
the prokaryotic proteins previously discussed. In contrast, RNA primers are
removed by RNase H and flap endonuclease 1 (FEN1) rather than by a DNA
polEukaryotic DNA Polymerases
5 High fidelity Eukaryotic DNA pols have been identified.
1. Pol α has a Primase activity that synthesizes a short RNA segment, which is
extended by polα 5’
3’ polymerase activity.
2. Pol ε and pol δ:
Pol ε is recruited to complete DNA synthesis on leading strand.
pol δ elongates okazaki fragments of the lagging strand.Eukaryotic DNA Polymerases
Each of them use 3’
5’ exonuclease activity to proofread newly
synthesized DNA.
3. Pol β and Pol γ: Pol β is involved in gap filling in DNA repair. Pol
γ replicates mitochondrial DNA.Proofreading newly synthesized DNA
DNA must be replicated with as few errors as possible.
Misreading of the template sequence could result in deleterious or
lethal mutations.
DNA Pol has a proofreading activity (3’ 5’ exonuclease) in addition
to its 5’
3’ polymerase activity.Proofreading newly synthesized DNATELOMERES & TELOMERASE
Telomeres are complexes of DNA plus Proteins located at the ends
of linear chromosomes.
They maintain the chromosomal integrity, preventing attack by
Nucleases.
23TELOMERES & TELOMERASE
Telomeres consists of several thousand tandem repeats of non
coding hexameric sequence “AGGGTT”.
Telomere shortening
Eukaryotic cells face a special problem during replication of the ends
of linear DNA molecules. Following removal of RNA primer from the
extreme 5’-end of the lagging strand.
2425TELOMERES & TELOMERASE
In most normal somatic cells, telomeres shorten with every cell
division.
Once it is shorter beyond a critical length, the cell is no longer able
to divide; “Senescence”.
Germ cells, stem cells& cancer cells: Telomers don’t shorten and
cells don’t senesce. This is a result of ribonucleoprotein
“Telomerase” which maintains the telomeric length in these cells.
26Telomerase
It is a complex containing a protein “Tert” that acts as a
Reverse Transcriptase and a short piece of RNA
“Terc” that acts as a template.
2728Reverse TranscriptionCentral Dogma• Normal transcription involves synthesis of RNA from DNA.
• Reverse transcription is the transcription of single stranded RNA into
double stranded DNA
• With the help of the enzyme Reverse Transcriptase.Retrovirus Replication Cycle ds DNA host DNA