Central Dogma

DNA Replication, Recombination, and Repair

Central Dogma

DNA Replication – process of producing identical copies of original DNA

• strand separation followed by copying of each strand

• fixed by base-pairing rules

DNA replication is bidirectional.

involves two replication forks that move in opposite direction

DNA replication requires unwinding of the DNA helix.

expose single-stranded templates

DNA gyrase – acts to overcome torsional stress imposed upon unwinding

helicases – catalyze unwinding of double helix- disrupts H-bonding of the two strands

SSB (single-stranded DNA-binding proteins) – binds to the unwound strands, preventing re-annealing

Primer

RNA primes the synthesis of DNA.

Primase synthesizes short RNA.

DNA replication is semidiscontinuous

DNA polymerase synthesizes the new DNA strand only in a 5’3’ direction. Dilemma: how is 5’ 3’ copied?

The leading strand copies continuously

The lagging strand copies in segments called Okazaki fragments (about 1000 nucleotides at a time) which will then be joined by DNA ligase

Overall: each of the two DNA duplexes contain one “old” and one “new” DNA strand (semi-conservative) and half of the new strand was formed by leading strand and the other half by lagging strand.

DNA Polymerase= enzymes that replicate DNA

All DNA Polymerases share the following:

1.Incoming base selected in the active site (base-complementarity)2.Chain growth 5’ 3’ direction (antiparallel to template)3.Cannot initiate DNA synthesis de novo (requires primer)First DNA Polymerase discovered – E.coli DNA Polymerase I (by Arthur Kornberg and colleagues)

Roger D. Kornberg

2006 Nobel Prize in Chemistry

Arthur Kornberg

1959 Nobel Prize in Physiology and Medicine

http://www.nobelprize.org

3’ 5’ exonuclease activity

- removes incorrect nucleotides from the 3’-end of the growing chain (proofreader and editor)- polymerase cannot elongate an improperly base-paired terminus

proofreading mechanisms• Klenow fragment – removes

mismatched nucleotides from the 3’’ end of DNA (exonuclease activity)

• detection of incorrect base- incorrect pairing with the template

(weak H-bonding)- unable to interact with the minor

groove (enzyme stalls)

DNA Ligase = seals the nicks between Okazaki fragments

DNA ligase seals breaks in the double stranded DNA

DNA ligases use an energy source (ATP in eukaryotes and archaea, NAD+ in bacteria) to form a phosphodiester bond between the 3’ hydroxyl group at the end of one DNA chain and 5’-phosphate group at the end of the other.

Eukaryotic DNA Replication Like E. coli, but more complex

Human cell: 6 billion base pairs of DNA to copy

Multiple origins of replication: 1 per 3000-30000 base pairs

E.coli 1 chromosomeHuman 23E.coli circular chromosome; Human linear

DNA Recombination =

recombinases Holliday junction – crosslike structure

natural process of genetic rearrangement

Mutations

1. Substitution of base pair

a. transitionb. transversion

2. Deletion of base pair/s

3. Insertion/Addition of base pair/s

DNA replication error rate: 3 bp during copying of 6 billion bp

Macrolesions: Mutations involving changes in large portions of the genome

Agents of Mutations

1. Physical Agentsa) UV Lightb) Ionizing Radiation

2. Chemical AgentsSome chemical agents can be

classified further intoa) Alkylatingb) Intercalatingc) Deaminating

3. Viral

UV Light Causes Pyrimidine Dimerization

Replication and gene expression are blocked

Chemical mutagens

• 5-bromouracil and 2-aminopurine can be incorporated into DNA

Deaminating agentsEx: Nitrous acid (HNO2)Converts adenine to hypoxanthine, cytosine to uracil, and

guanine to xanthineCauses A-T to G-C transitions

Alkylating agents

Intercalating agents

AcridinesIntercalate in DNA, leading to insertion or

deletionThe reading frame during translation is changed

DNA Repair

Direct repairPhotolyase cleave pyrimidine dimers

Base excision repairE. coli enzyme AlkA removes modified bases

such as 3-methyladenine (glycosylase activity is present)

Nucleotide excision repairExcision of pyrimidine dimers (need different

enzymes for detection, excision, and repair synthesis)

QUIZ1. Draw the structure of any nitrogenous base of your

picking. (1 pt)

2. What is the difference between the glycosidic bond and the phosphodiester bond? (2 pts)

3. Give the reason why DNA utilizes the deoxyribose while RNA uses the ribose. (2 pts)

4. Enumerate all the enzymes and proteins involved in DNA replication and briefly state their importance/function. A short concise answer will suffice. (4 pts)

5. Give the partner strand of this piece of DNA:5-ACTCATGATTAGCAG-3 (1 pt)

Central DogmaRNA Transcription

Process of Transcription has four stages:

1. Binding of RNA polymerase at promoter sites2. Initiation of polymerization3. Chain elongation4. Chain termination

Transcription (RNA Synthesis)

RNA PolymerasesTemplate (DNA)Activated precursors (NTP)Divalent metal ion (Mg2+ or Mn2+)

Mechanism is similar to DNA Synthesis

Reece R. Analysis of Genes and Genomes.2004. p47.

Limitations of RNAP II:

1. It can’t recognize its target promoter and gene. (BLIND)

2. It is unable to regulate mRNA production in response to developmental and environmental signals. (INSENSITIVE)

Start of Transcription

Promoter SitesWhere RNA Polymerase can indirectly bind

TATA box – a DNA sequence (5’—TATAA—3’) found in the promoter region of most eukaryotic genes.

Abeles F, et al. Biochemistry. 1992. p391.

Preinitiation Complex (PIC)

Transcription Factors (TF):

Hampsey M. Molecular Genetics of RNAP. Microbiology and Molecular Biology Reviews. 1998. p7.

TFIID binds to TATA; promotes TFIIB binding

TFIIA stabilizes TBP binding

TFIIB promotes TFIIF-pol II binding

TFIIF targets pol II to promoter

TFIIE stimulates TFIIH kinase and ATPase actiivities

TFII H helicase, ATPase, CTD kinase activities

Termination of Transcription

Terminator SequenceEncodes the

termination signalIn E. coli – base

paired hair pin (rich in GC) followed by UUU…

1. Intrinsic termination = termination sites

causes the RNAP to pause

causes the RNA strand to detach from the DNA template

Termination of Transcription

2. Rho termination = Rho protein, ρ

prokaryotes: transcription and translation happen in cytoplasm

eukaryotes: transcription (nucleus); translation (ribosome in cytoplasm)

In eukaryotes, mRNA is modified after transcriptionCapping, methylationPoly-(A) tailsplicing

capping: guanylyl residue

capping and methylation ensure stability of the mRNA template; resistance to exonuclease activity

Eukaryotic genes are split genes: coding regions (exons) and noncoding regions (introns)

Introns & Exons

IntronsIntervening

sequencesExons

Expressed sequences

Splicing

Spliceosome: multicomponent complex of small nuclear ribonucleoproteins (snRNPs)

splicing occurs in the spliceosome!

Central DogmaTranslation: Protein Synthesis

TranslationStarring three types of RNA

1.mRNA

2.tRNA

3.rRNA

Properties of mRNA

1. In translation, mRNA is read in groups of bases called “codons”

2. One codon is made up of 3 nucleotides from 5’ to 3’ of mRNA

3. There are 64 possible codons

4. Each codon stands for a specific amino acid, corresponding to the genetic code

5. However, one amino acid has many possible codons. This property is termed degeneracy

6. 3 of the 64 codons are terminator codons, which signal the end of translation

Genetic Code

3 nucleotides (codon) encode an amino acid

The code is nonoverlappingThe code has no punctuation

Synonyms

Different codons, same amino acidMost differ by the last base

XYC & XYU XYG & XYA

Minimizes the deleterious effect of mutation

Encoded sequences. (a) Write the sequence of the mRNA molecule

synthesized from a DNA template strand having the sequence

(b) What amino acid sequence is encoded by the following base sequence of an mRNA molecule? Assume that the reading frame starts at the 5 end.

Practice

A

Answers

(a) 5’ -UAACGGUACGAU-3’ .(b) Met-Pro-Ser-Asp-Trp-Met.

tRNA as Adaptor Molecules

Amino acid attachment site

Template recognition siteAnticodon

Recognizes codon in mRNA

tRNA as Adaptor Molecules

Mechanics of Protein Synthesis All protein synthesis involves three

phases: initiation, elongation, termination Initiation involves binding of mRNA and

initiator aminoacyl-tRNA to small subunit(30S), followed by binding of large subunit (50S) of the ribosome

Elongation: synthesis of all peptide bonds - with tRNAs bound to acceptor (A) and peptidyl (P) sites.

Termination occurs when "stop codon" reached

Translation: InitiationTranslation occurs in the ribosomeProkaryote START

fMet (formylmethionine) bound to initiator tRNA

Recognizes AUG and sometimes GUG (but they also code for Met and Val respectively)

AUG (or GUG) only part of the initiation signal; preceded by a purine-rich sequence

Translation: Initiation

Eukaryote START

AUG nearest the 5’ end is usually the start signal

Elongation

Termination

Stop signals (UAA, UGA, UAG):• recognized by release factors (RFs)• hydrolysis of ester bond between polypeptide and

tRNA

Reference:

Garrett, R. and C. Grisham. Biochemistry. 3rd edition. 2005.

Berg, JM, Tymoczko, JL and L. Stryer. Biochemistry. 5th edition. 2002.