DNA Replication Flashcards
- Discuss the meaning of these terms in the context of DNA Replication: “semi-conservative”, “bidirectional”, “Okazaki fragments”, “origin”, “fork”.
DNA replication is bidirectional and semiconservative.
- Bidirectional means that replication begins at a site named replication origin and simultaneously moves out in both directions from this origin.
- Eukaryotes have multiple replication origins on each chromosome
- Semiconservative means that, following replication, each newly replicated DNA double helix contains one intact parental strand and one newly synthesized daughter strand.
- *Replication forks** are the sites at which DNA synthesis is occurring.
- First, origin-binding proteins recognize and bind to origins of replication, which are AT rich sequences. The bacterial genome has one origin of replication, humans have 100s of origins of replication on each chromosome.
- 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 (SSB) bind to each single strand of DNA and hold it in a single-stranded conformation.
DNA polymerase is the key enzyme in replication and it synthesizes DNA in the 5’-to-3’ direction.
DNA polymerase 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. DNA Pol III is 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. DNA Pol I 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.
DNA Polimeraze need what to work:
1- A template strand (DNA)
2- It needs a primer, which is a RNA/DNA.
3- It needs a deoxynucleotides pool
4- Energy, which is released from a phosphoanhydride bond (high energy), to form a phosphodiester bond (low energy).
The bacterial genome has one origin of replication, humans have 100s of origins of replication on each chromosome.
Origin firing best understood in E.coli, which has a single origin with two different repeated sequences (yellow and blue in the diagram).
DnaA recognizes the blue sequences , it binds to it and forms an unwinding area near the yellow repeats, which allows DnaB (helicase) a and start unwinding the DNA at the origin, which allows primase and replicating enzymes to start they process.
Assembly of a pre-replication complex
In eukaryotes it is formed by six proteins (Origin Recognition Proteins) plus cdt1 and cdc6.
The pre-RC is not active since it happens in the G1 cycle of the cell, until it enters S phase of the cell, a kinase will activate it. Thus the cdt1 and cdc6 protein are regulators for timing of activation of the pre-RC.
Once these proteins are activated the DNA helicase (MCM) is loaded.
Steps in DNA replication
What protein protect the single-stranded DNA?
RPA (trimer) in humans and SSB in bacteria
-It binds the phospho-sugar backbone of DNA.
Distributive vs processive Polymerases
A distributive polymerase dissociates from DNA easily (DNA POL I in bacteria)
A processive polymerase holds on to DNA via a sliding clamp. (DNA POL III in bacteria)
- PCNA is the sliding clamp for eukaryotic DNA Pol d. It has a trimer structure and serves as a template for DNA replication, it allows the strand to rotate and other proteins to bind.
- b-subunit is the sliding clamp of prokaryotic DNA Pol III
*Clamp is loaded to DNA by clamp loader proteins
Comparison between E.coli polymerazes
Maturation of Okasaki fragments in E. Coli via DNA POL I
Maturation of Okasaki fragments in mammals
Major protein is FEN1: Flap structure-specific ENdonuclease
Lig1: DNA ligase 1
Pol d: DNA polymerase d (lagging strand polymerase)
DNA ligase seals the “nicks” between Okazaki fragments
Between 3’ OH and 5’ phosphate.
–>Uses energy from ATP
Accuracy of DNA Polymeraze
DNA polymerase discriminate between correct and incorrect nucleotides on the basis of:
-Hydrogen bonds that specify correct pairing between complimentary bases
-Common geometry of the A-T and G-C base pairs that is required to fit in the active site of the polymerase.
DNA polymerases incorporate one incorrect nucleotide per 104 ~ 105 correct ones.
Proofreading activity of DNA polymerase lowers error rate to 10-6 ~ 10-8
The polarity problem
For bacteria
Comparison between bacteria and eukaryotes
DNA pol E is the main polymeraze for the leading strand,
DNA pol D is the polymeraze for the lagging strand, and DNA POL A serves a role in development of the primer (aids primase activity).
Comparisson of replisome component
Overview of DNA replication in eukaroytes
Replication of retroviral genome - conversion of viral ssRNA to dsDNA by “reverse transcription”
Reverse transcription
- Synthesis of DNA from an RNA template is catalyzed by reverse transcriptase.
- Retroviruses contain RNA as their genetic material. The retroviral RNA serves as a template for synthesis of DNA by reverse transcriptase.
- Telomerase has a reverse transcriptase activity and carries its own RNA template, in order to restore the ends of chromosomes (telomeres) in human cancer and stem cells.
Replication machineries of pathogens as therapeutic targets
The “end-replication problem”
The “end replication problem” 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.
- Telomerase is an RNA-dependent DNA polymerase that maintains chromosomal ends by copying the telomeric repeat sequence from an RNA template. Telomerase activity is repressed in normal somatic cells.
- In cancer cells, the telomerase enzymeis 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.
Bypass of telomere shortening in cancer cells
DNA replication and human disease
Rare recessive mutant alleles of genes encoding DNA replication proteins cause inherited genetic disorders that are often characterized by reduced growth, premature ageing and increased cancer risk.
Cancerous cells often have increased expression levels of DNA replication proteins, which can be used as clinical screening and prognosis biomarkers.
Therapeutic drugs have been developed to target DNA replication for cancer treatment.
Mutations in genes encoding replication proteins have been found in inherited human genetic disorders that are often characterized with premature aging, reduced growth, and increased risk of cancer. e.g. Mutations in RecQ helicases BLM and WRN cause Bloom Syndrome and Werner’s Syndrome, respectively, characterized by premature aging, increased cancer risk and cardiovascular problems. Mutations in the pre-replication complex cause Meier-Gorlin syndrome (dwarfism).
- Cellular levels of some DNA replication proteins such as MCM5 can be sensitive biomarkers for early detection and prognosis of many common cancers.
- DNA replication-targeting drugs have been developed as cancer therapeutics.
RecQ helicase mutations cause progeric syndromes
pre-RC mutations cause Meier-Gorlin syndrome (dwarfism)
DNA replication proteins as biomarkers in cancer screening
DNA replication-targeting therapeutics
