dna replication semi-conservative mechanism 1958, meselson & stahl 15 n labeling experiment

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  • DNA replicationSemi-conservative mechanism1958, Meselson & Stahl

    15N labeling experiment

  • Rosalind Franklin (1920-1958) Maurice Wilkins (1916-2004)Francis Crick (1916-2004)James Watson (1928-)Discovery of DNA structure 1962 Nobel Prize

  • The substrates of DNA synthesis

    dNTPs dATP, dGTP, dCTP, dTTP

    Direction: 5-3

  • ++ ppi 5PPP5PPP5 3

  • 3 5 ???

  • ???

  • ppProofreading???

  • Replicon is any piece of DNA which replicates as a single unit. It contains an origin and sometimes a terminus

    Origin is the DNA sequence where a replicon initiates its replication.Terminus is the DNA sequence where a replicon usually stops its replication

  • All prokaryotic chromosomes and many bacteriophage and viral DNA molecules are circular and comprise single replicons.

    There is a single termination site roughly 180o opposite the unique origin.

  • The long, linear DNA molecules of eukaryotic chromosomes consist of mutiple regions, each with its own orgin.A typical mammalian cell has 50000-100000 replicons with a size range of 40-200 kb. When replication forks from adjacent replication bubbles meet, they fuse to form the completely replicated DNA. No distinct termini are required

  • Semi-discontinuous replicationExperimental evidences [3H] thymidine pulse-chase labeling experiment

    1. Grow E. coli2. Add [3H] thymidine in the medium for a few second, spin down and break the cell to stop labeling, analyze and find a large fraction of nascent DNA (1000-2000 nt) = Okazaki fragments3. Grow the cell in regular medium then analyze, the small fragments join into high molecular weight DNA = Ligation of the Okazaki fragments

  • Back

  • Bacterial DNA replicationExperimental systems

    1. Purified DNA: smaller and simpler bacteriophage and plasmid DNA molecules (X174, 5 Kb)2. All the proteins and other factors for its complete replications

  • Study system

    the E. coli origin locus oriC is cloned into plasmids to produce more easily studied minichromosomes which behave like E.coli chromosome.

    Initiation: oriC

  • 1. oriC contains four 9 bp binding sites for the initiator protein DnaA. Synthesis of DnaA is coupled to growth rate so that initiation of replication is also coupled to growth rate.2. DnaA forms a complex of 30-40 molecules, facilitating melting of three 13 bp AT-rich repeat sequence for DnaB binding.3. DnaB is a helicase that use the energy of DNA hydrolysis to further melt the double-stranded DNA .4. Ssb (single-stranded binding protein) coats the unwinded DNA.5. DNA primase attaches to the DNA and synthesizes a short RNA primer for synthesis of the leading strand.6. Primosome DnaB helicase and DNA primase

  • Unwinding

    Positive supercoiling: caused by removal ofhelical turns at the replication fork.Resolved by a type II topoisomerase calledDNA gyrase

  • ElongationDNA polymerase III holoenzyme

    1. A dimer complex, one half synthesizing the leading strand and the other lagging strand.2. Having two polymerases in a single complex ensures that both strands are synthesized at the same rate3. Both polymerases contain an -subunit---polymerase -subunit---3 5 proofreading exonuclease -subunit---clamp the polymerase to DNA other subunits are different.

  • Replisome

    in vivo DNA polymerase holoenzyme dimer, primosome (helicase) are physically associated in a large complex to synthesize DNA at a rate of 900 bp/sec.

    Other two enzymes during Elongation

    1. Removal of RNA primer, and gap filling with DNA pol I 2. Ligation of Okazaki fragments are linked by DNA ligase.Prokaryotic DNA replication

  • Termination and segregationTerminus containing several terminator sites (ter) approximately 180o opposite oriC.Tus protein ter binding protein, an inhibitor of the DnaB helicaseTopoisomerase IV a type II DNA topoisomerase, function to unlink the interlinked daughter genomes.

  • Eukaryotic DNA replicationExperimental systems

    1. Small animal viruses (simian virus 40, 5 kb) are good mammalian models for elongation (replication fork) but not for initiation.2. Yeast (Saccharomyces cerevisiae): 14 Mb in 16 chromosomes, 400 replicons, much simpler than mammalian system and can serve as a model system3. Cell-free extract prepared from Xenopus (frog) eggs containing high concentration of replication proteins and can support in vitro replication.

  • Cell cycle

  • Entry into the S-phase

    CyclinsCDKs (Cyclin-dependent protein kinases)

  • DNA Replication

    DNA replication is semi-conservative, one strand serves as the template for the second strand. Furthermore, DNA replication only occurs at a specific step in the cell cycle. The following table describes the cell cycle for a hypothetical cell with a 24 hr cycle.

    Stage Activity Duration G1 Growth and increase in cell size 10 hr S DNA synthesis 8 hr G2 Post-DNA synthesis 5 hr M Mitosis 1 hr

    DNA replication has two requirements that must be met:

    1.DNA template 2.Free 3' -OH group

  • Origin and initiation1. Clusters of about 20-50 replicons initiate simultaneously at defined times throughout S-phase Early S-phase: euchromatin replication Late S-phase: heterochromatin replication Centromeric and telomeric DNA replicate last

    2. Only initiate once per cell cycle Licensing factor required for initiation inactivated after use can only enter into nucleus when the nuclear envelope dissolves at mitosis

  • Electron Microscopy of replicating DNA revealsreplicating bubbles.

  • 3. Individual yeast replication origins (ARS) have been cloned into prokaryotic plasmids which allow these plasmids to replicate in yeast (an eukaryote). ARSs autonomously replicating sequences Minimal sequence 11 bp [A/T]TTTAT[A/G]TTT[A/T] (TATA box)

    4. ORC (origin recognition complex) binds to ARS, upon activation by CDKs, ORC will open the DNA for replication.

  • Elongation1. Replication fork

    - unwinding DNA from nucleosomes: 50 bp/sec - need helicases and replication protein A (RP-A) - new nucleosomes are assembled to DNA from a mixture of old and newly synthesized histones after the fork passes

  • 2. Elongation

    Three different DNA polymerases are involved

    1) DNA pol contains primase activity and synthesizes RNA primers for the leading strands and each lagging strand fragments. Continues elongation with DNA but is replaced by the other two polymerases quickly.2) DNA pol on the leading strand that replaces DNA pol ., can synthesize long DNA3) DNA pol on the lagging strand that replaces DNA pol ., synthesized Okazaki fragments are very short (135 bp in SV40), reflecting the amount of DNA unwound from each nucleosome.

  • Nuclear matrix1. A scaffold of insoluble protein fibers which acts as an organizational framework for nuclear processing, including DNA replication, transcription

    2. Replication factories containing all the replication enzymes and DNA associated with the replication forks in replicationBudR labeling of DNA

  • Telomere replication

  • Telomerase

    1. Contains a short RNA molecule as telomeric DNA synthesis template2. Telomerase activity is repressed in the somatic cells of multicellular organism, resulting in a gradual shortening of the chromosomes with each cell generation, and ultimately cell death (related to cell aging)3. The unlimited proliferative capacity of many cancer cells is associated with high telomerase activity.

  • Telomerase activity is repressed in somatic cells of multicelluar organisms resulting in a gradual shortening of the chromosome with each cell generation. As this shortening reaches informational DNA, the cells senesce and die. cell divisioninformational DNAWhen telomerase activity is repressed

  • MutagenesisMutation

    Permanent, heritable alterations in the base sequence of DNA

    Reasons1. Spontaneous errors in DNA replication or meiotic recombination2. A consequence of the damaging effects of physical or chemical mutagens on DNA

  • Point mutationA singe base change: transition, transversion

    The effects of point mutation Phenotypic effectsNoncoding DNANonregulatory DNA Silent mutation No 3rd position of a codon

    Coding DNA altered AA Missense mutation Yes or No

    Coding DNA stop codon Nonsense mutation Yes Truncated protein

  • Insertions & deletions The addition or loss of one or more bases in a DNA region

    Frameshift mutations The ORF of a protein encoded gene is changed so that the C-terminal side of the mutation is completely changed.

    Genetic polymorphisms Caused by accumulation of many silent and other nonlethal mutations

  • Replication fidelityImportant for preserve the genetic information from one generation to the next, spontaneous errors in DNA replication is very rare, e.g. one error per 1010 base in E. coli.

    Molecular mechanisms for the replication fidelity1. DNA polymerase: Waston-Crick base pairing2. 3 5proofreading exonuclease.3. RNA priming: proofreading the 5end of the lagging strand4. Mismatch repair

  • MutagensCausing DNA damage that can be converted to mutations.

    Physical mutagens High-energy ionizing radiation X-rays and -rays strand breaks and base/sugar destruction Nonionizing radiation UV light pyrimidine dimersChemical mutagens Base analogs direct mutagenesis Nitrous acid deaminates C to produce U Alkylating agents Arylating agents indirect-lesion mutagenesis Intercalators: e.g. EB

  • MutagenesisThe molecular process in which the mutation is generated.

    Note the great majority of lesions introduced by chemical and physical mutagens are repaired by one or more of the error-free DNA repair mechanisms before the lesions is encounter by a replication fork

    Direct mutagenesis The stable, unrepaired base with altered base pairing properties in the DNA is fixed to a mutation during DNA replication.

  • Indirect mutagenesis The mutation is introduced as a result of an error-prone repair. Translesion DNA synthesis to maintain the DNA integrity but not the sequence accuracy when damage occurs immediately ahead of an advancing fork, which is unsuitable for recombination repair, the daughter strand is synthesized regardless of the the base identity of the damaged sites of the parental DNA.

  • DNA damage and repairDNA lesionsOxidative damage1. Occurs undernormal condition2. Increased byionizing radiationphysical mutagens

    AlkylationAlkylating agentsChemical mutagensBulky adductsUV lightphysical mutagensCarcinogenChemical mutagens

  • Biological effects of the unrepaired DNA lesions

    Physical distortion ofthe local DNA structure

    Blocks replicationand/or transcription

    LethalAltered chemistry of the bases

    Allowed to Remain in the DNA

    A mutation could become fixed by direct or indirect mutagenesis

    Mutagenic

  • Spontaneous DNA lesions1. Inherent chemical reactivity of the DNA2. The presence of normal, reactive chemical species within the cell - Deamination C U methylcytosine T - Depurination break of the glycosylic bond, non-coding lesion - Depyrimidine

  • Oxidative damage1. occurs under NORMAL conditions in all aerobic cells due to the presence of reactive oxygen species (ROS), such as superoxide, hydrogen peroxide, and the hydroxyl radicals (OH).

    2. The level of this damage can be INCREEASED by hydroxyl radicals from the radiolysis of H2O caused by ionizing radiation

  • Alkylation1. Electrophilic chemicals adds alkyl groups to various positions on nucleic acids

    2. Distinct from those methylated by normal methylating enzymes.

    3. Typical alkylating agents: MMS methylmethane sulfonate EMS ethylmethane sulfonate ENU ethylnitrosourea

  • Bulky adducts1. DNA lesions that distort the double helix and cause localized denaturation, for example pyrimidine dimers arylating agents adducts

    2. These lesions disrupt the normal function of the DNA

  • DNA repair

    Photoreactivation

    1. Monomerization of cyclobutane pyrimidine dimers by DNA photolyases in the presence of visible light

    2. Direct reversal of a lesion and is error-free

  • Alkyltransferase

    1. Removing the alkyl group from mutagenic O6-alkylguanine which can base-pair with T. The alkyl group is transferred to the protein itself and inactivate it.

    2. Direct reversal of a lesion and is error-free

    3. In E.coli, The response is adaptive because it is induced by low levels of alkylating agents and gives increased protection against the lethal and mutagenic effects of the high doses

  • Excision repair

    1. Including nucleotide excision repair (NER) base excision repair (BER)

    2. Ubiquitous mechanism repairing a variety of lesions.

    3. Error-free repair

  • Nucleotide excision repair (NER)1. An endonucleasecleaves DNA a precisenumber of bases onboth sides of the lesions(e.g. in E.coli, UvrABCEndonulcease removespyrimidine dimers)2. Excised lesion-DNAfragment is removed3. The gap is filled byDNA polymerase Iand sealed by ligase

  • Base excision repair (BER)

  • A specialized form of excision repair whichdeals with any base mispairs producedduring replication and which have escapedproofreading

    Mismatch repair

  • The parental strand is methylated at N6 position of all As in GATC sites, but methylation of the daughter strand lag a few minutes after replicationMutH/MutS recognize the mismatched base pair and the nearby GATCDNA helicase II, SSB, exonuclease Iremove the DNA fragment including the mismatchDNA polymerase III & DNA ligase fill in the gap

  • Essay questions1. How to explain the mechanisms of semi-conservative replication and semi-discontinuous replication? How to verify them by experiments?

    2. How about the differences between prokaryotic and eukaryotic DNA replication?

    3. How about the main types of DNA damage? and the main repair mechanisms?

  • DNA recombination- Homologous recombination- Site-specific recombination- TranspositionAn important reason for variable DNA sequencesamong different populations of the same species

  • Homologous recombination1. Homologous duplicated chromosomes line up in parallel in metaphase I.2. The nonsister chromatids exchange equivalent sections by crossing over. The exchange of homologous regions between two DNA moleculesIn diploid eukaryotes, it commonly occurs during meiosis

  • Crossing over

  • Haploid prokaryotes recombination- between the replicated portions of a partially duplicated DNA- between the chromosomal DNA and acquired foreign DNA, like plasmids or phagesOccurs between the two homologous duplex

  • Nick formation

  • RecA-ssDNA filament

  • Recombination-based DNA repair

  • Site-specific recombination1. Exchange of non-homologous but specific pieces of DNA

    2. Mediated by proteins that recognize specific DNA sequences.

  • Bacteriophage insertion1. -encoded integrase (Int): makes staggered cuts in the specific sites2. Int and IHF (integration host factor encoded by bacteria): recombination and insertion3. -encoded excisionase (XIS): excision of the phage DNA

  • Antibody diversityH and L are all encoded by three gene segments: V, D, J

    V D JTwo heavy chains (L) 250 15 5Two light chains (H) 250 4Enormous number (>108) of different H and L gene sequences can be produced by such a recombination

  • Transposition1. Requires no homology between sequences nor site- specific

    2. Relatively inefficient

    3. Require Transposase encoded by the transposon

  • TransposonsE. coli - IS elements/insertion sequence 1-2 kb, comprise a transposase gene flanked by a short inverted terminal repeats Tn transposon series carry transposition elements and -lactamase (penicillin resistance)

  • Eukaryotic transposons many are retrotransposons

    Yeast Ty element encodes protein similar to RT(reverse transcriptase)