Molecular Biology

BBM FK UNTAR

Flow of Genetic Information in the Cell• Mechanisms by which information is transferred

in the cell is based on “Central Dogma”

The central dogma of molecular biology. Solid arrows indicate the types of genetic information transfers that occur in all cells. Special transfers are indicated by the dashedarrows: RNA-directed RNA polymerase is expressed both by certain RNA viruses and by some plants; RNA-directed DNA pol (reverse transcriptase) is expressed by other RNA viruses; and DNA directly specifying a protein is unknown but does not seem beyond the realm of possibility. However, themissing arrows are information transfers the central dogma postulates never occur: protein specifying either DNA, RNA, or protein. In other words, proteins can only be the recipients of genetic information.

1869 Friedrich Miescher, precipitate nuclein Hoppe-Seyler isolated a similar substance from yeast cells – RNA

1944 Oswald Avery, Colin MacLeod, and Maclyn McCarty - DNA is the molecule that carries genetic information.1953 James D.Watson and Francis H. C. Crick proposed their model for the structure of double-stranded DNA.1956 Meselson & Sthal : Replication is Semiconservative1957, Arthur Kornberg, et al discovered DNA polymerase I. 1960, Erwin Chargaff : A = T and G = C for each species1961, Marshall Nirenberg & J. Heinrich Matthaei , discovered poly U, code genetic1965 Nirenberg and H. Gobind Khorana, the rest of the code was deciphered1975 F Sanger DNA Sequencing

History

1.Nucleotides Are the Building Blocks of Nucleic Acids

2. DNA Is Double-Stranded- A. Nucleotides Are Joined by 3 –5 Phosphodiester Linkages- B. Two Antiparallel Strands Form a Double Helix- C. Weak Forces Stabilize the Double Helix (4 type)- D. Conformations of Double-Stranded DNA (3)

- Most of the DNA in a cell is negatively supercoiled

3. DNA Can Be Supercoiled

4. Cells Contain Several Kinds of RNA5. Nucleosomes and Chromatin6. Nucleases and Hydrolysis of Nucleic Acids

Chemical structures of the major pyrimidines and purines

DNA base pairing

Chargaff’s rule s

A = TC = G

A diagrammatic representation of the Watson and Crick model of the double-helical structure of the B form of DNA

The relationship between the sequences of an RNA transcript and its gene

1. Nucleic acids are polymers of nucleotides that are phosphate esters of nucleosides. The amino and lactam tautomers of the bases form hydrogen bonds in nucleic acids.

2. DNA contains two antiparallel strands of nucleotide residues joined by 3 –5 PDE linkages. A and G in one strand pair with T and C, respectively/

3. The double-helical structure of DNA is stabilized by hydrogen bonding, hydrophobic effects, stacking interactions, and charge–charge interactions. G/C-rich DNA is more difficult to denature than A/T-rich DNA because the stacking interactions of G/C base pairs are greater than those of A/T base pairs.

4. The most common conformation of DNA is called B-DNA; alternative conformations include A-DNA and Z-DNA.

NUCLEIC ACIDS

5. Overwinding or underwinding the DNA helix can produce supercoils that restore the B conformation. Negatively supercoiled DNA exists in equilibrium with DNA that has locally unwound regions.

6. The four major classes of RNA are rRNA, tRNA, mRNA, and sRNA. RNA molecules are single stranded and have extensive secondary structure.

7. Eukaryotic DNA molecules are packaged with histones to form nucleosomes. Further condensation and attachment to the scaffold of a chromosome achieves an overall 8000-fold reduction in the length of the DNA molecule in metaphase chromosomes.

8. The phosphodiester backbones of nucleic acids can be hydrolyzed by the actions of nucleases. Alkaline hydrolysis and RNase A catalyzed hydrolysis of RNA proceed via a 2 ,3 -cyclic nucleoside monophosphate intermediate.

9. Restriction endonucleases catalyze hydrolysis of DNA at specific palindromic nucleotide sequences. Specific methylases protect restriction sites from cleavage.

10. Restriction enzymes are useful for constructing restriction maps of DNA, for DNA fingerprint analysis, and for constructing recombinant DNA molecules.

DNA Replication

DNA Replication Since genetic information is carried in DNA, the transfer of information from a

parental cell to two daughter cells requires exact duplication of DNA, a process known as DNA replication.

The process of DNA replication is complex and involves many cellular functions and several verification procedures to ensure fidelity in replication.

Meselson and Stahl (1958) DNA Replication Is semiconservative

DNA Replication Is Bidirectional DNA replication. (1) Initiation, (2) Elongation and (3) Termination DNA Polymerase :

- A. Chain Elongation Is a Nucleotidyl Group Transfer Reaction- B. DNA Polymerase III Remains Bound to the Replication Fork- C. Proofreading Corrects Polymerization Errors: 3-5 exonuclease

DNA Polymerase Synthesizes Two Strands Simultaneously: - DNA pol catalyze chain elongation exclusively in the 5 – 3 direction, - Lagging Strand Synthesis Is Discontinuous - Each Okazaki Fragment Begins with an RNA Primer - Okazaki Fragments Are Joined by the Action of DNA Pol I and DNA Ligase

Steps Involved in DNA Replication in Eukaryotes

1. Identification of the origins of replication2. Unwinding (denaturation) of dsDNA to provide an ssDNA template3. Formation of the replication fork; synthesis of RNA primer4. Initiation of DNA synthesis and elongation5. Formation of replication bubbles with ligation of the newly synthesized DNA segments6. Reconstitution of chromatin structure

DNA polymerases Deoxynucleotide polymerizationHelicases Processive unwinding of DNATopoisomerases Relieve torsional strain that results from helicase-

induced unwindingDNA primase Initiates synthesis of RNA primersSingle-strand binding proteins (SSB) Prevent premature reannealing of dsDNADNA ligase Seals the single strand nick between the nascent

chain and Okazaki fragments on lagging strand

Protein Function

Prokaryotic vs. eukaryotic DNA replication

Prokaryotes:

• Single origin of DNA replication (circular chromosome)

Eukaryotes:

• Multiple origins of DNA replication

• Occurs during S phase of the cell cycle

Diagram of the replication fork. The two newly synthesized strands have opposite polarity. On the leading strand, synthesis moves in the same direction as the replication fork; on the lagging strand, synthesis moves in the opposite direction.

DNA replication is “semi-conservative”

(Figure obtained at www.sparknotes.com)

DNA replication:

1) Separation of the two strands

2) Complete replication using each strand as a template for the synthesis of a new “daughter” strand

DNA replication is also bi-directional

• Replication starts at “origins of replication” or “replication fork”

• Can have multiple origins within a chromosome - efficient

DNA polymerase: the enzyme that makes DNA

• Many more DNA polymerases

Eukaryotes:

• Five DNA polymerases: I, II, III, IV, and V

• Can be broadly categorized into replication or repair

Prokaryotes (E. coli):

Substrate: deoxyribonuceloside triphosphate

Common structure of DNA polymerase

Enzyme has independent domains

Conserved sequence motif for catalytic active site

Responsible for positioning template correctly at the active site

Binds DNA as it exits the enzyme

Process of DNA replicatiton

1. Helicase separates both strands of the DNA

2. Single-stranded proteins bind and maintain separated strands

3. Prime with 3’-OH end (difference between leading & lagging strand)

4. Synthesis of DNA by DNA polymerase

5. Ligation of Okazaki fragments by ligase (lagging strand only)

(Figure obtained at Ohio State Biosci website)

A different view of DNA replication process

Things to also consider: chromatin access by trans-factors

The discontinuous polymerization of deoxyribonucleotides on the lagging strand; formation of Okazaki fragments during lagging strand DNA synthesis is illustrated. Okazaki fragments are 100–250 nt long in eukaryotes, 1000–2000 bp in prokaryote

The DNA Polymerase Complex

Three important properties: (1) chain elongation, (2) processivity, and (3) proofreading

DNA pol III associates with the two identical subunits of the DNA sliding "clamp"; this association dramatically increases pol III-DNA complex stability, processivity (10 ntd to >50,000 ntd) and rate of chain elongation (20 to 50 ntd/sec) generating the high degree of processivity the enzyme exhibits.

Polymerase I (pol I) and II (pol II) are mostly involved in proofreading and DNA repair. Eukaryotic cells have counterparts for each of these enzymes plus a large number of additional DNA pol primarily involved in DNA repair.

DNA polymerase structures more or less follow a common architectural pattern that is reminiscent of a right hand, with distinctstructural domains referred to as fingers, palm, and thumb

The more recently discovered eukaryoticDNA polymerases (including ή ζ ι κ and Rev1) are novel in that they aremore error-prone, resulting in lower fidelity of DNA replication. Nevertheless, they have the important ability to function in DNA replication and repair when damaged regions of DNA are encountered.

Other proteins involved in eukaryotic DNA replication include replicationprotein A (RPA), an ssDNA-binding protein that is the eukaryotic counterpartof SSB, and replication factor C (RFC). RFC loads the PCNA sliding clamp onto replicating DNA, thus acting as the eukaryotic counterpart of the prokaryotic γ-complex.

Model of the ReplisomeThe replisome contains a primosome, the DNA polymerase III holoenzyme, and additional proteins that are required for DNA replication. The assembly of many proteins into a single machine allows coordinated synthesis of the leading and lagging strands at the replication fork.

Accesories proteins : - helicase (DnaB)- topoisomerase II (gyrase)- single-strand binding protein (SSB) -

Model of the Replisome

The replisome contains a primosome, the DNA polymerase III holoenzyme, and additional proteins that are required for DNA replication.

Diagram of the subunit composition of E. coli DNA polymerase III. The holoenzyme consistsof two core complexes (containing a, e and u), paired copies of b and t, and a single g complex (g, d, d , with two copies each of c,and x). The structure is thus an asymmetric dimer. Other models of the holoenzymestructure have been proposed.

DNA Polymerase Synthesizes Two Strands Simultaneously:

- A. Lagging Strand Synthesis Is Discontinuous

- B. Each Okazaki Fragment Begins with an RNA Primer

- C. Okazaki Fragments Are Joined by the Action of DNA Polymerase I and DNA Ligase

DNA polymerase I contains the two activities found in the DNApolymerase III holoenzyme: 5-3 polymerase activity and 3-5 proofreading exonuclease activity. In addition, DNA polymerase I has 5-3 exonuclease activity, an activity not found in DNA polymerase III.

DNA polymerase I can be cleaved with certain proteolytic enzymes to generate a small fragment that contains the 5-3 exonuclease activity and a larger fragment that retains the polymerization and proofreading activities.

The larger fragment consists of the C-terminal 605 amino acid residues, and the smaller fragment contains the remaining N-terminal 323 residues.

The large fragment, known as the Klenow fragment, was widely used for DNA sequencing and is still used in many other techniquesthat require DNA synthesis without degradation. In addition, many studies of the mechanisms of DNA synthesis and proofreading use the Klenow fragment as a model for more complicated DNA polymerases.

The unique 5-3 exonuclease activity of DNA polymerase I removes the RNA primer at the beginning of each Okazaki fragment

(a) Completion of Okazaki fragment synthesis leaves a nick between the Okazaki fragment and the preceding RNA primer on the lagging strand.

(b) DNA polymerase I extends the Okazaki fragment while its 5 →3 exonuclease activity removes the ′ ′RNA primer. This process, called nick translation, results in movement of the nick along the lagging strand.

(c) DNA polymerase I dissociates after extending the Okazaki fragment 10–12 nucleotides.DNA ligase binds to the nick.

(d) DNA ligase catalyzes formation of aphosphodiester linkage, which seals the nick, creating a continuous lagging strand. The enzyme then dissociates fromthe DNA.

(a) The lagging-strand template loops back through the replisome so that the leading and lagging strands are synthesized in the same direction. SSB binds to single-stranded DNA

(b) As helicase unwinds the DNA template, primase synthesizes an RNA primer. The lagging-strand polymerase completes an Okazaki fragment

Simultaneous synthesis ofleading and lagging strands at a replication fork.

The replisome contains the DNA polymerase III holoenzyme (only the core complexes are shown);a primosome containing primase, a helicase, and other subunits; and additional components including SSB.

One core complex of the holoenzymesynthesizes the leading strand while the other core complex synthesizes the lagging strand.

The lagging-strand templateis looped back through the replisome so that the leading and lagging strands can be synthesized in the same direction as fork movement.

(c) When the lagging-strand polymerase encounters the preceding Okazaki fragment, it releases the lagging strand.

(d) The lagging-strand polymerase binds to a newly synthesized primer and beginssynthesizing another Okazaki fragment.

Initiation and Termination of DNA Replication

DNA sequence called the origin. In E. coli, this site is called oriC, and it is located at about 10 o’clock on the genetic map of the chromosome.

The initial assembly of replisomes at oriC depends on proteins that bind to this site causing local unwinding of the DNA.

DnaA helps regulate DNA replication by controlling the frequency of initiation. The initial RNA primers required for leading-strand synthesis are probably made by the primosomes at the origin.

Termination of replication in E. coli occurs at the termination site (ter), a regionopposite the origin on the circular chromosome. This region contains DNA sequencesthat are binding sites for a protein known as terminator utilization substance (Tus).

Tus prevents replication forks from passing through the region by inhibiting the helicase activity of the replisome. The termination site also contains DNA sequences that play a role in the separation of daughter chromosomes when DNA replication is completed.

DNA Replication in Eukaryotes

DNA Repair

• Spontaneous DNA damage: spontaneous alteration of bases, depurination and deamination, thymine dimer

• Pathways to remove DNA damage: base excision repair, nucleotide excision repair

• Damage detection: base flipping• The repair of Double-strand break:

nonhomolous end joining, homologous end joining

• DNA repair enzymes: heat shock proteins

Types of Damage to DNA

I. Single-base alteration

A. DepurinationB. Deamination of cytosine to uracilC. Deamination of adenine to hypoxanthineD. Alkylation of baseE. Insertion or deletion of nucleotideF. Base-analog incorporation

II. Two-base alterationA. UV light–induced thymine-thymine (pyrimidine) dimerB. Bifunctional alkylating agent cross-linkage

III. Chain breaksA. Ionizing radiationB. Radioactive disintegration of backbone elementC. Oxidative free radical formation

IV. Cross-linkageA. Between bases in same or opposite strandsB. Between DNA and protein molecules (eg, histones)

Mechanism of DNA RepairMismatch repairCopying errors (single base or two- to five-base unpaired loops)Methyl-directed strand cutting, exonuclease digestion, and replacement

Base excision–repairSpontaneous, chemical, or radiation damage to a single baseBase removal by N -glycosylase, abasic sugar removal, replacement

Nucleotide excision–repairSpontaneous, chemical, or radiation damage to a DNA segmentRemoval of an approximately 30-nucleotide oligomer and replacement

Double-strand break repairIonizing radiation, chemotherapy, oxidative free radicalsSynapsis, unwinding, alignment, ligation

Mismatch repair Copying errors (single base or two- Methyl-directed strand cutting, to five-base unpaired loops) exonuclease digestion and

replacement

Base excision repair Spontaneous, chemical, or radiation Base removal by N –glycosylase, damage to a single base abasic sugar removal,

replacement

Nucleotide excision – Spontaneous, chemical, or radiation Removal of an approximately repair damage to a DNA segment 30-nucleotide oligomer and replacement

Double-strand break- Ionizing radiation, chemotherapy, Synapsis, unwinding, alignment,Repair oxidative free radicals ligation.

Mechanism Problem Solution