Structure&Fuction of NT

a. initially incorporates 1 wrong nucleotide sequence every ~106 bp

b. overall accuracy is much higher at 1 error per 109 bp

DNA polymerase reverses its direction by one base pair of DNA.

• The 3’-5’ exonuclease activity of the enzyme allows the incorrect
• base pair to be excised.

Following this excision, the polymerase can re-insert the correct base and replication can continue.

• 1. bidirectional and semi-conservative
• 2. Synthesis only in the 5’ to 3’ direction
• 3. requires processive and distributive polymerases
• 4. requires RNA primers on both strands near origin of replication5. highly dependent on Watson-Crick base paring

RNase H, DNA ligase, DNA polymerase δ, single-stranded DNA binding proteins (SSBs), topoisomerases, helicase, primase, replication protein A (RPA), FEN1

proofreading of polymerases and DNA repair system

• 1. RNA primer hybridizes to ssDNA (synthesized by primase or RNA polymerase)
• 2. elongation by a DNA polymerase, generating Okazaki fragments
• 3. when the okazaki fragment encounters the next RNA primer, RNase activity (or 5’ to 3’ exonuclease activity by polymerase I in prokaryotes) removes the primer (additional FEN1 in eukaryotes)
• 4. gap filling (by distributive polymerase)
• 5. nick sealing (by DNA ligase

• 1 Enzymes (ex: DNA helicase, DNA
• Polymerase, RNase, DNA Topoisomerase) in order to bypass this problem.

• 2 AT because they are easier to break
• since they have only two hydrogen bonds whereas GC possesses three hydrogen bonds.

1. short fragment of DNA created on the lagging strand during DNA replication

2. synthesis is initiated from an RNA primer at the 5’ terminus

DNA repair (gap filling); has 5'->3' activity and both 3'->5' exonuclease(Proofreading) and 5'->3' exonuclease activity (RNA Primer removal)

involved in reparation of damaged DNA; has 3'->5' exonuclease activity

main polymerase in bacteria (elongates in DNA replication); has 3'->5'exonuclease proofreading ability

cleaving nucleotides one at a time from the end of a polynucleotide chain. A hydrolyzing reaction occurs that breaks phosphodiester bonds at either the 3’ or 5’ ends.

DNA repair and replication

1. links together two DNA strands that have double-strand breaks (a break in both complementary strands of DNA)

2. fixes a single-stranded break where it uses the complementary strand as a template but requires the ligase to create the final phosphodiester bond for full repair.

1. unwind/wind DNA to control the synthesis of proteins & DNA replication

2. untwist the parental strand of DNA (transesterification).

transient breaks in one strand

breaks in both strands

a. essential in bacteria for replication and drug target for antibiotics

b. essential in humans as a target for cancer chemotherapy

• 1. DNA polymerase I contains a 5' exonuclease that clips off the RNA primer contained immediately upstream from the site of DNA synthesis in a 5' → 3' manner
• 2. the 5' -> 3' exonuclease activity makes it unsuitable for many applications
• a. can be removed from the holoenzyme to leave a useful molecule (Klenow fragment)

• 1. processive enzyme: used in the sense of going in a forward direction (5’->3’) and continues down this path without coming unbound, as in DNA Polymerase III.
• Ex: polymerase that carries out many additions of nucleotide b4 dissociating from DNA
• 2. distributive enzyme: catalyzes one cleavage first and then another one on another chain at random (gap filling), as in DNA Polymerase I.

• 1. region of repetitive DNA at the end of a chromosome
• 2. protects the end of the chromosome from deterioration
• 3. “caps” the end of the chromosome, protecting it from homologous recombination and non-homologous end joining.

• a. intermediate in homologous genetic recombination
• b. important in maintaining genomic integrity
• c. exists in prokaryotes and eukaryotes

• double-stranded (duplex) molecule of nucleic acid originated through the genetic recombination of single complementary strands derived from different sources, such as from different homologous chromosomes
• Ex: Regions where one strand was from one duplex, and the other from the homologous duplex.

• sequences of DNA that can move around to different positions
• a. process called transposition catalyzed by transposases
• b. two types: simple and replicative transposition
• c. can cause mutations and change the amount of DNA in the genome
• d. insertion sequences possess inverted repeats on both ends
• e. they are flanked by direct repeats (same orientation) in the largest sequence

methyl guanine methyl transferase (MGMT). The reaction is stoichiometric rather than catalytic

mismatch repair if a wrong base is incorporated during replication

• cellular mechanism that repairs damaged DNA throughout the cell cycle. It is primarily responsible for removing small, non-helix distorting base lesions from the genome.
• i. removal of damaged base by specific glycosidases
• ii. repair of AP sites left after removal of a damaged base (or by spontaneous hydrlysis)
• iii. AP endonucleases/AP lyases excise a basic sugar, gap filling, ligation

• prevent unwanted mutations by removing the vast majority of photodimers
• i. recognize bulky distortions in the shape of the DNA double helix
• ii. endonucleases excise a stretch of nucleotides
• iii. gap-filling in by DNA polymerase I
• iv. ligation by DNA ligase
• v. mismatch: if wrong base is incorporated but is undamaged
• vi. transcription-coupled: lesions block transcription but transcribed regions are repaired faster

endonuclease and a DNA ligase

a. damage tolerance process that allows the DNA replication machinery to replicate past DNA lesions such as thymine dimers or AP sitesb. involves switching out regular DNA polymerases for specialized translesion polymerases (e.g. DNA polymerase V),

• i. piece of daughter strand replaces damaged portion of parental strand
• ii. normal DNA duplex then repairs daughter strand
• iii. normal DNA duplex replicates missing portion given to daughter strand
• iv. most likely to occur on lagging strand

• i. results because of ROS or topoisomerase inhibitors
• ii. can be homologous (yeast) or nonhomologous end joining (mammalian cells)

• i. recA binds to ssDNA regions at blocked replication forks to maintain integrity
• ii. fork regression by branch migration (in protected state)

• iii. lesion repair by base or nucleotide expansion repair
• iv. reversal of branch migration

• a. induced upon extensive DNA damage (SOS reponse)
• b. readily dissociates from DNA (distributive process so low processivity)
• c. ensure continued replication if a lesion is encountered by the replication machinery
• d. error prone and lack proofreading ability
• e. induced to prevent a long halt of replication at lesions

A, T, G, C (in RNA, U replaces T)

• a. 1 polymerase
• b. specific σ factors recognize class of genes
• ex) σ factor binds to RNA polymerase core enzyme
• c. RNA polymerase binds to the promoter region first

a. 3 polymerase

b. transcription factors bind to DNA sequences

c. transcription factors (or TATA binding protein (TBP)) binds to the promoter region first

• d. require transcription factors but not σ factors for transcription
• i. specific transcription factors recognize gene-specific DNA sequences
• ii. transcription factors are proteins that oftentimes form dimers

• i. Pribnow (AT-rich)
• ii. -35 region (conserved and asymmetrical)

i. “open” complex from middle of Pribnow box to around start site (-9 to +2)

ii. easier for AT than GC (Pribnow box now AT-rich)


iii. increase of GC pairs would decrease promoter efficiency

iv. increase of AT pairs would INCREASE promoter efficiency

• 1. RNA strand is read from 3’ to 5’
• 2. RNA is synthesized (transcribed) in the 5’ to 3’ direction

1. RNA polymerase does NOT require a primer

a. RNA polymerase molecules can transcribe one gene simultaneously

2. DNA polymerase DOES require a primer

1. Daunomycin and Adriamycin

2. inhibits initiation of RNA transcription

• 1. doesn’t have to be as accurate as that of DNA
• a. genetic code degenerate (errors may be silent)
• b. quick degradation and re-synthesis (high turnover rate)
• c. one AA substitution in protein is oftentimes without consequence

• 2. no repair system known
• a. degradation and re-synthesis
• b. high turnover rate

• 1. Prokaryotes: occurs because prokaryotic mRNA does not need to be transported or processed, therefore, translation can begin immediately after transcription
• 2. Eukaryotes: does not occur because mRNA must be processed and transported before translation can take place

1. Prokaryotes: protein translation often occurs with little or no modification

2. Eukaryotes: extensive modification is possible

1. 5’-terminal cap (7-methyl G)

2. 3’-terminal poly A tail

3. methylated internal variants

4. splice variants

• 1. modification of RNA after transcription
• 2. excision of intron sequences (noncoding) and exons are joined
• 3. needed before eukaryotic mRNA can be used for translation
• 4. involves several transesterification processes
• 5. involves the formation of a 2’,5’-phosphodiester bond
• 6. requires Watson-Crick base pairing with other RNA molecules for high accuracy

RNA polymerase I carries out the transcription in eukaryotes3. in

prokaryotes, transcription factors are not involved in rRNA transcription

• 1. they have a length of approximately 21-23 nucleotides
• 2. they are double-stranded
• 3. they inhibit gene expression by degrading homologous mRNA
• 4. function (posttranscriptional) gene splicing (degradation of homologous mRNA sequences)

• Important drug targets for treatment of microbial diseases and/orparasitic diseases. Sites for manipulation of biological systems mutagenesis studies preparation of antibodies
• Biological processes where genetic alterations have severe and far-reaching consequences

IMP

DNA polymerase needs primers while RNA polymerase does not

Top: energy for chain elongation would come from hydrolysis of triphosphate of chain

Bottom: removal of a wrong paired 5’-terminal nucleotide triphosphate during proof reading would prevent further chain elongation

RNA fragments as primers (8-10 nt in humans; synthesized by primase or RNA polymerase) for synthesis of Okazaki fragments

• RNase Hybridase
• DNA Polyermase
• DNA Ligase

• Task: holding DNA polymerase in contact with DNA
• Allows DNA polymerase to be more processive
• (executes many reactions before dissociating

pol III, main, processive

(pol I, gap filling, distributive) + 3’ → 5’ proofreading exonuclease activity + 5’ → 3’ exonuclease activity (RNase H; primer removal)

In bacteria: Topoisomerase II is essential for replication and drug target for antibiotics

In humans: Topoisomerase II is essential: target for cancer chemotherapy

Eukaryotes (more DNA!) Thousands of origins (every 3-300 kb, tissue- and species-dependent)

Prokaryotes one single ori

• Termination requires topoisomerase II
• bidirectionaly

1. S phase of cell cycle


2. G1- (longest phase of growth) or S cell cycle arrest

Exchange of genetic info. can be homologous (identical sequence) or non homologous

A: Look at methylation pattern! Higher degree of methylation in parental strand

extensive DNA damage (SOS response) and readily dissociates from DNA (distributive)

• Putative mechanism:
• 1. RecA binds to ssDNA regions at blocked replication forks to maintain integrity
• 2. Fork regression by branch migration (in protected state)
• 3. Lesion repair by base or nucleotide excision repair
• 4. Reversal of branch migration

• 1.Initiation recognition of DNA sequence by RNA polymerase
• 2.Elongation RNA chain synthesis
• 3.Termination release of RNA chain/RNA polymerase from DNA template

Prokaryotes: 1 polymerase + specific σ factors recognize classes of genes

Eukaryotes: 3 polymerases + transcription factors bind to DNA sequences

1. DNA protected from alkylation (dimethyl sulfate) where enzyme is bound → Pribnow and -35 region

2. Certain nitrogens usually involved in H-bonding only gets alkylated if ssDNA → "open" complex from middle of Pribnow box to ca. start site (-9 to +2)


Easier for A:T than for G:C (Note: Pribnow box is AT rich)

Doesn not require primer

Tolerable precision

1 wrong base per 104 transcribed bases -genetic code degenerate -one aa substitution in protein often w/out consequences


Genes can be transcribed by several RNA polymerase molecules at the same time

Ribosomal peptide synthesis from mRNA matrix

1.Amino acid code unchanged

2.Stop codon

3.Change of amino acid code

-insertion/deletion

three tRNA binding sites: A, P, E

1.Initiation

2.Elongation

3.Termination

4.Posttranslational Modification