Molecular Biology: DNA Replication & Repair

DNA Damage and Repair

• DNA replication achieves high accuracy with only about one error per 10 billion nucleotides.
• Base pairing specificity is crucial for this accuracy.
• Proofreading and error-checking mechanisms ensure near-perfect matching of base pairs during replication.
• Other enzymes further correct errors left behind by DNA polymerase or created after DNA synthesis.

DNA Proofreading and Repair

• DNA polymerase proofreads each nucleotide against its template immediately after bonding.
• If a mistake is found, the wrong nucleotide is replaced with the correct one.
• DNA polymerase has exonuclease abilities to remove incorrect nucleotides.

Nucleotide Excision Repair (NER)

• Reactive chemicals, radioactivity, X-rays, ultraviolet light, and other harmful chemicals can damage DNA.
• Mismatched nucleotides can sometimes evade DNA polymerase proofreading.
• Mismatch repair removes and replaces incorrectly paired nucleotides.
• Repair mechanisms include nucleases, DNA polymerase, and DNA ligase.

DNA Mismatch Repair

• Parental DNA is methylated, daughter DNA is not.
• Repairing errors is crucial for organism survival.
• Specific enzymes catalyze DNA repair.

UV-Related DNA Damage in Skin Cells

• UV light covalently crosslinks two adjacent pyrimidines, most commonly creating thymine dimers.
• This distorts the DNA molecule.
• Repair machinery detects the distortion and initiates the appropriate repair pathways.

Repair: UV Damage

• Xeroderma pigmentosum (XP) is an inherited disease involving defects in nucleotide excision repair mechanisms, causing extreme sensitivity to sunlight.
• Mutations in skin cells from uncorrected UV damage often lead to skin cancer.
• Without sun protection, children with XP can develop skin cancer by age 10.

DNA Packaging

• Stretched out, E. coli DNA would be about a millimeter long, 500 times longer than the cell.
• Proteins cause the chromosome to coil and supercoil, packing the DNA densely.
• Unlike eukaryotic cells, the dense DNA region called the nucleoid in prokaryotes is not enclosed in a membrane.

DNA Packaging: Eukaryotes

• Each eukaryotic chromosome contains a single linear DNA double helix.
• If stretched out, a human chromosome would be approximately 4 cm long.
• Eukaryotic DNA is precisely combined with a large amount of protein, forming chromatin.

DNA Packaging: Chromatin

• Chromatin undergoes changes in its degree of packing during the cell cycle.
• Interphase chromatin is highly extended.
• Prophase chromatin coils/folds (condenses)
• Metaphase chromosomes are short and thick.

Chromatin: Spatial Organization

• Looped domains of chromatin are attached to the nuclear envelope during interphase.
• These attachments organize regions of active chromatin.
• Chromatin of each chromosome occupies a specific area within the nucleus.

Heterochromatin vs Euchromatin

• Some chromatin regions are more/less condensed than others.
• Heterochromatin is highly condensed, DNA largely inaccessible to transcriptional machinery.
• Euchromatin is less tightly packed, DNA in these regions is more easily transcribed, usually associated with actively transcribed genes.
• Chemical modifications of histones influence chromatin condensation and gene activity.

Transcription: Gene → Protein

• DNA inherited by an organism dictates protein synthesis and RNA molecule synthesis.
• This produces specific traits of an organism.
• Proteins link genotype and phenotype.
• Gene expression is the process of DNA directing the synthesis of proteins (or, in some cases, just RNAs).

Individual Genes Specify Individual Enzymes

• Neurospora has modest food requirements.
• It can grow in minimal nutrients solution incorporated into agar medium
• Neurospora uses the minimal media and its own metabolic pathways to produce needed molecules.
• Experiments by Beadle and Tatum demonstrated a one-gene-one-enzyme model. Each enzyme is encoded by a specific gene.

One Gene - One Enzyme

• Beadle and Tatum bombarded Neurospora cells with X-rays to generate mutants needing arginine.
• They isolated mutants unable to grow in minimal medium without arginine into three classes.
• This demonstrated that each mutant was defective in one gene and produced one specific enzyme.

One Gene - One Enzyme: Flaws & Revisions

• Not all genes encode enzymes.
• Many genes encode proteins without enzymatic properties.
• Many proteins are constructed from multiple polypeptide chains.
• A more accurate model is one gene - one polypeptide.
• Many eukaryotic genes code for a set of closely related polypeptides via alternative splicing.
• Some genes code for RNA molecules not translated into proteins.

Transcription & Translation

• Genes have hundreds or thousands of nucleotides.
• Each polypeptide of a protein has amino acids arranged in a specific linear order (primary structure).
• Transcription synthesizes RNA using information in DNA.
• Different RNA types can be produced via transcription.
• Translation synthesizes a polypeptide using information in mRNA.
• Translation changes the language from nucleic acids to amino acids.

Transcription & Translation (Bacteria vs Eukaryotes)

• Bacteria lack nuclear membranes, thus translation can begin before transcription is finished.
• Eukaryotic transcription occurs in the nucleus, producing pre-mRNA that subsequently undergoes post-transcriptional modification before translation in the cytoplasm.
• All types of initial RNAs are transcribed (primary transcripts).

Transcription

• RNA nucleotides match with the DNA template strand sequence.
• RNA polymerase synthesizes RNA in the 5' to 3' direction.

Gene Components

• Prokaryotic genes have regulatory sequences (including promoter) and coding regions (exons).
• Eukaryotic genes have regulatory sequences (including promoter), coding regions (exons), and noncoding regions (introns).
• Transcribing into an RNA molecule is called the transcription unit.

Steps of Transcription

• Transcription involves RNA polymerase.
• RNA polymerase synthesizes in the 5' to 3' direction.
• RNA polymerase does not require a primer to begin.
• Prokaryotes have one type of RNA polymerase, while eukaryotes have three or more.
• Transcription occurs in initiation, elongation, and termination steps.