DNA Replication , DNA Damage and Repair
OutlineCentral Dogma()DNA Replication()Testing Models for DNA replicationSemi-conservative replication()Evidence for Semi-Discontinuous() ReplicationE. coli DNA polymerasesDetails of DNA Replication Three steps 1) Initiation 2) Elongation 3) Termination and Separation Eukaryotic DNA Replication Like E. coli, but more complex DNA Damage and RepairTypes of Repair
DNARNAProtein Central Dogma
Central DogmaTranscriptionTranslationReplicationReplicationRetro-transcriptionGene expressionCentral Dogma
transcription:Protein synthesis is referred to as translation. gene expression):Gene expression is the process by which a gene's information is converted into the structures and functions of a cell. The process in which DNA is used to synthesize RNA is called transcription.translation):Central Dogma
transcription: mRNA,,mRNAgene expression):DNARNAtranslation):
Coding strand, Sense strand, Crick strandTemplate strand, antisense strand, Watson strandTranscriptionTranslationCentral Dogma
Happy Birthday, Double Helix
DNA ReplicationBackground Information Watson & Crick Double-helix Structural Model
In 1953, James Watson and Francis Crick deduced the SecondaryStructure of DNADouble-helix Structural Model () . This was one of the most important biological advances, since it led to an understanding of the relationship of the DNA structure to its function, particularly to the way it was replicated (). DNA Replication
"Molecular Structure of Nucleic Acids: A Structure for Deoxyribose Nucleic Acid" (Nature, April 25, 1953. volume 171:737-738.) "...It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material. The structure itself suggested that each strand could separate and act as a template for a new strand, therefore doubling the amount of DNA, yet keeping the genetic information, in the form of the original sequence, intact. "DNA Replication
Can you remember The Basic Features of B-DNA?
Can you remember The Basic Features of B-DNA?There have 5 features and 6 parameters. DNA Replication
4. Bases are Restricted Pairing on the InsideA purine-pyrimidine pair fits PERFECTLY. Three hydrogen bonds hold the deoxyguanosine nucleotide to the deoxycytidine nucleotide, whereas the other pair, the A-T pair, is held together by two hydrogen bonds. The Basic Features of B-DNA
5. A Pair of Chains are Complementary SequencesThe bases are complementary, with A on one side of the molecule you only get T on the other side, similarly with G and C. If we know the base sequence of one strand we know its complement. The Basic Features of B-DNA
DNA Replication General FeaturesDNA Replication
DNA Replication General Features 1) Many enzymes and proteins are required
2) Template & dNTPs/Mg 2+ are required
3) Semi-conservative ( A key experiment designed by M. Meselson and W. F. Stahl 1958
4) DNA Unwinding is necessary
5) A Primer ( with a free 3' -OH group is required
DNA Replication General Features6) Only in the 53direction
7) Specific Origin of Replication-Ori C()
8) Bi-directional (With some exceptions)
9) Semi-discontinuous () Replication fork () Leading strand (), Lagging strand () and Okazaki fragments ()
10) Highly processive (), Highly ordered and Extremely accurate
Testing Models for DNA replicationMatthew Meselson and Franklin Stahl (1958)DNA Replication
Matthew Meselson and Franklin Stahl more recentlyFaculty member at HarvardMechanisms of Molecular EvolutionFaculty Chair for CBW StudiesFaculty member at U. of OregonMeiotic RecombinationDNA Replication
1958MeselsonStahlDNA DNA Replication
Density labeling experiment on E. coli (bacterial) DNADNA Replication
Meselson and Stahl (continued)Harvest some cells1st generationHarvest some cells2nd generationFor each generation isolate the DNA and spin through a density (CsCl) gradient).Detect DNA in the gradient (by UV absorption)
Monitor how many DNA bands there are after each generation
Meselson and Stahl Original DataDNA Replication
DNA ReplicationSemi-conservative replication?
Semi-conservative replication: After the two strands separate, each serves as a template for the synthesis of a complementary strand.(In other words, each of the two new DNA molecules contains one old strand and one new strand.) This process, referred to as semiconservative replication. DNA Replication
Since DNA replication is semiconservative, therefore the helix must be unwound.DNA Replication
John Cairns (1963) showed that initial unwinding is localized to a region of the bacterial circular genome, called an origin or ori for short.
John CairnsCairns then isolated the chromosomes by lysing the cells very very gently and placed them on an electron micrograph (EM) grid which he exposed to X-ray film for two months.DNA Replication
Evidence points to bidirectional replicationDNA Replication
DNA Replication is Semi-discontinuousConsider one replication fork:Continuous replicationDiscontinuous replicationDNA Replication
DNAreplication forkDNA Replication
Evidence for the Semi-Discontinuous replication model was provided by the Okazakis (1968)DNA Replication
Evidence for Semi-Discontinuous Replication(pulse-chase experiment) Harvest the bacteriaat different timesafter the chaseIsolate their DNASeparate the strands(using alkali conditions)Run on a sizing gradientRadioactivity will onlybe in the DNA that was made during the pulseDNA Replication
Results of pulse-chase experiment DNA Replication
Continuous synthesisDiscontinuous synthesisDNA replication is semi-discontinuousDNA Replication
Many enzymes and proteins are required in DNA Replication.
Many enzymes and proteins are required in DNA Replication.
Do you know how many enzymes are required in DNA Replication?
TopoisomeraseDNA HelicaseDNASingle-stranded DNA binding proteins SSBPrimase- formation of RNA primersDNA dependent DNA polymerase III (DNA pol, DNAIII The Enzymes responsible for removing RNA primers (DNA polymerase I) DNA ligase DNA -joining of Okazaki fragmentsEnzymes and Proteins Involved in DNA ReplicationDNA Replication
E. coli DNA polymerasesIdentification Kornberg and DNA pol I (Kornberg enzyme)Structure and Function of DNA pol I A multi-functional enzymeDNA pol III is a major polymerase involved in E. coli chromosome DNA replicationDNA Replication
DNA polymerase Ipol I1956KornbergEcoliDNADNA1959Kornberg DNA Replication
DNA Pol I from E. coli is 928 aa (109 kD) monomer A single polypeptide with at least three different Enzymatic activities! How Amazing!!!
a 3 to 5 exonuclease activity a 5 to 3 exonuclease activity a 5 to 3 DNA polymerizing activity
Proof reading activityof the 3 to 5 exonuclease.
Proof reading activity is slowcompared to polymerizingactivity, but the stalling ofDNAP I after insertion of an incorrect base allows the proofreading activity to catch up with the polymerizingactivity and remove theincorrect base.DNA Replication
A total of 5 different DNAPs have been reported in E. coli DNAP I: does 90% of polymerizing activity DNAP II: functions in DNA repair (proven in 1999)DNAP III: principal DNA replication enzyme DNAP IV: functions in DNA repair (discovered in 1999)DNAP V: functions in DNA repair (discovered in 1999)The DNA Polymerase Family DNA Replication
The "real" replicative polymerase in E. coli Its accurate: makes 1 error in 107 dNTPs added, with proofreading, this gives a final error rate of 1 in 1010 overall. fast: up to 1,000 dNTPs added/sec/enzyme Its highly processive: >500,000 dNTPs added before dissociatingDNA Polymerase III ITS COMPLICATED!!!DNA Replication
The structure formed by two beta subunits of the E. coli DNA polymerase III . This structure can clamp a DNA molecule and slide with the core polymerase along the DNA molecule. DNA Replication
DNA Polymerase IIIholoenzyme ReplicationForkLeading Strand synthesisLagging Strand synthesisDNA Replication
pol Ipol IIpol III103Kd88Kd900Kd1 7 104001001010.05155'3'3'5'5'3' RNA5'3'
DNA Polymerase III is principal DNA replication enzyme in DNA replication.
But the other important Enzymes and Proteins Involved in DNA Replication? DNA Replication
Action of DNA LigaseDNA Replication
NAD+:E+NAD+EAMP+NMNDNA ATP: E+ATPEAMP+ppi
DNA Replication is an Ordered Series of Steps.
The Following is Details of DNA Replication.DNA Rep