Part 3 Genetic Information Transfer. The biochemistry and molecular biology department of CMU. Central dogma. replication. transcription. translation. DNA. protein. RNA. reverse transcription. Replication: synthesis of daughter DNA from parental DNA
Genetic Information Transfer
The biochemistry and molecular biology department of CMU
Section 1General Concepts of DNA Replication
Daughter strand synthesis
Phosphodiester bond formation
DNA replication system
Template: double stranded DNA
Primer: short RNA fragment with a free 3´-OH end
Enzyme:DNA-dependent DNA polymerase (DDDP),
§1.1 Semi-Conservative Replication
Half of the parental DNA molecule is conserved in each new double helix, paired with a newly synthesized complementary strand. This is called semiconservative replication
The genetic information is ensured to be transferred from one generation to the next generation with a high fidelity.
Replication starts from unwinding the dsDNA at a particular point (called origin), followed by the synthesis on each strand.
The parental dsDNA and two newly formed dsDNA form a Y-shape structure called replication fork.
§1.2 Bidirectional Replication
Once the dsDNA is opened at the origin, two replication forks are formed spontaneously.
These two replication forks move in opposite directions as the syntheses continue.
Replication of prokaryotes
The replication process starts from the origin, and proceeds in two opposite directions. It is named replication.
Chromosomes of eukaryotes have multiple origins.
The space between two adjacent origins is called the replicon, a functional unit of replication.
Replication of eukaryotes
origins of DNA replication (every ~150 kb)
The daughter strands on two template strands are synthesized differently since the replication process obeys the principle that DNA is synthesized from the 5´ end to the 3´end.
§1.3 Semi-continuous Replication
On the template having the 3´- end, the daughter strand is synthesized continuously in the 5’-3’ direction. This strand is referred to as the leading strand.
Many DNA fragments are synthesized sequentially on the DNA template strand having the 5´- end. These DNA fragments are called Okazaki fragments. They are 1000 – 2000 nt long for prokaryotes and 100-150 nt long for eukaryotes.
The daughter strand consisting of Okazaki fragments is called thelagging strand.
Continuous synthesis of the leading strand and discontinuous synthesis of the lagging strand represent a unique feature of DNA replication. It is referred to as thesemi-continuous replication.
Section 2Enzymology of DNA Replication
§2.1 DNA Polymerase
DNA-pol of prokaryotes
1. 53 polymerizing
DNA-pol of E. coli
α： has5´→ 3´ polymerizing activity
ε：has3´→ 5´ exonuclease activity and plays a key role to ensure the replication fidelity.
θ: maintain heterodimer structure
DNA-pol of eukaryotes
DNA-pol : initiate replication and synthesize primers
DNA-pol : replication with low fidelity
DNA-pol : polymerization in mitochondria
DNA-pol : elongation
DNA-pol : proofreading and filling gap
Also called DnaG
Primase is ableto synthesize primers using free NTPs as the substrate and the ssDNA as the template.
Primers are short RNA fragments of a several decades of nucleotides long.
Primers provide free 3´-OH groups to react with the -P atom of dNTP to form phosphoester bonds.
Primase, DnaB, DnaC and an origin form a primosomecomplex at the initiation phase.
Also referred to as DnaB.
It opens the double strand DNA with consuming ATP.
The opening process with the assistance of DnaA and DnaC
§2.4 SSB protein
Opening the dsDNA will create supercoil ahead of replication forks.
The supercoil constraint needs to be released by topoisomerases.
Topoisomerase I (topo I)
Also called -protein in prokaryotes.
It cuts a phosphoester bond on one DNA strand, rotates the broken DNAfreely around the other strand to relax the constraint,and reseals the cut.
Topoisomerase II (topo II)
It is named gyrase in prokaryotes.
It cuts phosphoester bonds on both strands of dsDNA, releases the supercoil constraint, and reforms the phosphoester bonds.
It can change dsDNA into the negative supercoil state with consumption of ATP.
§2.6 DNA Ligase
Connect two adjacent ssDNA strands by joining the 3´-OH of one DNA strand to the 5´-P of another DNA strand.
Sealing the nick in the process of replication, repairing, recombination, and splicing.
§2.7 Replication Fidelity
Proofreading and correction
5´→3´ exonuclease activity
cut primer or excise mutated segment
3´→5´ exonuclease activity
excise mismatched nuleotides
Section 3DNA Replication Process
§3.1 Replication of prokaryotes
Genome of E. coli
Structure of ori C
Formation of preprimosome
Formation of replication fork
Releasing supercoil constraint
Lagging strand synthesis
§3.2 Replication of Eukaryotes
telomerase association protein
telomerase reverse transcriptase
Significance of Telomerase
Section 4Other Replication Modes
§4.1 Reverse Transcription
The genetic information carrier of some biological systems is ssRNA instead of dsDNA (such as ssRNA viruses).
The information flow is from RNA to DNA, opposite to the normal process.
This special replication mode is called reverse transcription.
Reverse transcription is a process in which ssRNA is used as the template to synthesize dsDNA.
Process of Reverse transcription
Synthesisof ssDNA complementary to ssRNA, forming a RNA-DNA hybrid.
Hydrolysis ofssRNA in the RNA-DNA hybrid by RNase activity of reverse transcriptase, leaving ssDNA.
Synthesis of the second ssDNA using the left ssDNA as the template, forming a DNA-DNA duplex.
Reverse transcriptase is the enzyme for the reverse transcription. It has activity of three kinds of enzymes:
RNA-dependent DNA polymerase
DNA-dependent DNA polymerase
Significance of RT
An important discovery in life science and molecular biology
RNA plays a key role just like DNA in the genetic information transfer and gene expression process.
RNA could be the molecule developed earlier than DNA in evolution.
RT is the supplementary to the central dogma.
Significance of RT
This discovery enriches the understanding about the cancer-causing theory of viruses. (cancer genes in RT viruses, and HIV having RT function)
Reverse transcriptase has become a extremely important tool in molecular biology to select the target genes.
§4.2 Rolling Circle Replication
§4.3 D-loop Replication
Section 5DNA Damage and Repair
Mutation is a change of nucleic acids in genomic DNA of an organism. The mutation could occur in the replication process as well as in other steps of life process.
§5.2 Causes of Mutation
Mutation caused by chemicals
§5.3 Types of Mutation
a. Point mutation (mismatch)
Point mutationis referred to as the singlenucleotide alternation.
Transition:the base alternation from purine to purine, or from pyrimidine to pyrimidine.
Transversion: the base alternation between purine and pyrimidine, and vise versa.
Hb mutation causing anemia
Singlebase mutation leads to one AA change, causing disease.
b.Deletion and insertion
Deletion: one or more nucleotides aredeleted from the DNA sequence.
Insertion: one or more nucleotides are inserted into the DNA sequence.
Deletion and insertion can cause the reading frame shifted.
5´… …GCAGUACAUGUC … …
Ala Val His Val
5´… …GAGUACAUGUC … …
Glu Tyr Met Ser
It is an exchange of large DNA fragments. It can be either reverse the direction or recombination between chromosomes.
1. Site-specific recombination
2. Homologous genetic recombination
3. DNA transposition
§5.4 DNA Repairing
One of the most important and effective repairing approach.
UvrA and UvrB: recognize and bind the damaged region of DNA.
UvrC: excise the damaged segment.
DNA-pol Ⅰ: synthesize the DNA segment to fill the gap.
DNA ligase: seal the nick.
Xeroderma pigmentosis (XP)
XP is an autosomal recessive genetic disease. Patients will be suffered with hyper-sensitivity to UV which results in multiple skin cancers.
The cause is due to the low enzymatic activity for the nucleotide excision-repairing process, particular thymine dimer.